Precision in Action: The Role of Clustered Regularly Interspaced Short Palindromic Repeats/Cas in Gene Therapies.

1. Introduction

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein (Cas) exists extensively in archaea and bacteria as an innate immune system. The rapid development of the CRISPR/Cas genome editing tools has revolutionized the gene therapy field and enhanced our ability to treat various genetically heritable diseases [ 1 ]. CRISPR was first identified by scientists while analyzing a gene for alkaline phosphatase in the prokaryotic Escherichia coli genome [ 2 , 3 ]. The sequence consisted of 29 nucleotide repeats broken up by 32 nucleotide spacer sequences that were only observed while the bacteria were in contact with exogenous DNA [ 3 , 4 ]. Prior to applying CRISPR systems to gene therapy, these unique repeats were exclusively used as a biomarker throughout the process of genotyping [ 5 ]. The Discovery of CRISPR across several bacterial and archaeal genomes led to the isolation of CRISPR-adjacent genes (Cas1-Cas4) that play a large role in our understanding of this mechanism’s editing ability [ 6 , 7 ]. The lactic acid bacteria Streptococcus thermophilus was later used to analyze the relationship between CRISPR and Cas proteins, revealing their coupled ability to provide prokaryotes with an advanced immune system for protection against bacteriophages and viruses [ 8 ].

2. Mechanism of CRISPR/Cas’s Action

CRISPR sequences exist in various classes and species, containing different repeats and involved genes [ 9 , 10 ]. All types of CRISPR-Cas systems have been classified based on utilizing multi-subunit Cas-protein complexes (class I) or a single versatile Cas-protein (class II) [ 11 ]. The different types of CRISPR are described in Table 1 . Studies have shown that class I CRISPR/Cas contains types I, II, and IV [ 12 ]. The system of interest in modern medicine and gene therapy is the class II CRISPR-Cas system, which includes type II, V, and VI [ 12 ]. The CRISPR-Cas9 and CRISPR-Cas12a effectors are type II and type V, respectively [ 13 , 14 , 15 ]. Type VI includes CRISPR-Cas 13 and is used for RNA editing [ 16 ]. Class 2, type V includes miniature CRISPR-Cas 12J, CRISPR-Cas 12f, and CRISPR-associated transposase [ 17 , 18 , 19 ]. Studies have shown that type I CRISPR-Cas are newly emerging tools for transcriptome and genome manipulation in eukaryotic cells [ 20 , 21 ] and microbiota [ 22 ]. The two essential components of the original Cas9 system from Streptococcus pyogenes (spCas9) include guide RNA (gRNA) and the CRISPR-associated Cas9 protein (1368 amino acids) [ 23 ]. The editing mechanism can be broken down into three phases: recognition, cleavage, and repair [ 24 ]. Recognition involves specialized gRNA capable of directing the Cas9 protein and detecting the genomic target sequence. The Cas9 nuclease acts as a scissor, creating a double-stranded break (DSB) at several nucleotides upstream of a complementary “protospacer-adjacent motif” (PAM) sequence [ 23 ]. Figure 1 illustrates the mechanism of CRISPR action and its application in different cellular processes. Cas9 is unable to cleave DNA in the absence of PAM. Structurally, the Cas9 nuclease consists of two active domains, RuvC and HNH (His-Asn-His), utilized for non-complementary and complementary DNA cleavage, respectively [ 25 ]. Cas9 contains a bilobed structure, an REC lobe (recognition lobe), and an NUC lobe (nuclease lobe). The REC lobe recognizes and binds to gRNA, and the NUC lobe consists of RuvC, HNH, and PAM interacting domains responsible for binding to target DNA [ 25 ]. The gRNA consists of CRISPR RNA (CrRNA) and trans-activating CRISPR RNA (TracrRNA). CrRNA contains a spacer sequence that recognizes target DNA by base pairing, while TracrRNA contains several loops to form a binding scaffold structure for Cas9 interaction. CrRNA and TracrRNA form single guide RNA (sgRNA) via an artificial linker [ 26 , 27 ]. SpCas9 recognizes the 5′-NGG-3′ sequence as a PAM in the target DNA [ 28 , 29 ]. Cas9 creates a DSB in target sequence [ 30 ] at a site 3 bp away from PAM. Following DSB, the DNA fragment of the bacteriophage or plasmid is expelled from the local genome, subsequently providing the host organism with immunity against invading mobile genetic elements. In the repair phase, non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways repair DSB lesions via host cell machinery [ 31 ]. DNA repair in the NHEJ process is mediated by inserting or deleting DNA base pairs. NHEJ is an error-prone repair pathway. An error can occur at any cell cycle phase, leading to insertion/deletion (InDel) mutations in target DNA [ 32 , 33 ]. In the presence of an appropriate sister chromatid donor, HDR allows for precise insertion or deletion, thus, error-free DNA repair ( Figure 1 ). The HDR is most active in the late S2 and G2 phases of the cell cycle. The CRISPR/Cas system has recently been used for epigenome editing, prime editing, and base editing. Base editors are mainly classified into two classes: the adenine base editor (AD) and the cytosine base editor (CD) ( Figure 1 ). The use of the CAS system in the cytosine base editor was reported in 2016 and has been used in many studies for base editing (A-T to G-C) in genomes [ 34 , 35 , 36 , 37 ]. Many studies have employed the CAS system as an adenine base editor (G-C to T-A) [ 38 , 39 , 40 ]. All types of transition mutations (G to A, T to C, C to A, and A to G) can be inserted into the genome using these AD and CD base editors [ 41 ]. The base editors can also be engineered to induce specific C to G transversion [ 42 ]. An engineered CAS, which is inactivated due to the insertion of mutations called dCAS [ 43 ], can be fused with a transcription factor to regulate gene expression [ 44 , 45 ], a repressor to repress the gene expression [ 43 ], a DNA modifying enzyme or histone modifying enzyme to perform epigenome editing [ 46 , 47 ], and genome imaging [ 48 , 49 ].

3. CRISPR and Other Genome Editing Methods

Since the discovery of CRISPR, several adaptations have been made to its molecular mechanism to enhance practices within biomedicine, agriculture, and others [ 5 ]. Through many clinical trials, CRISPR technology has been proven to have higher rates of efficacy for gene editing when compared to older gene therapy systems, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [ 4 , 53 ]. ZFN and TALEN tools rely on restriction enzymes and guide proteins, whose engineering is difficult, time consuming, and expensive. ZFNs are transcription factors that bind specifically to DNA sequences, and a new ZFN needs to be engineered for each new DNA targeted, which is a laborious and difficult process. TALENs are made from the fusion of a TALE (transcription activator-like effector) and the catalytic domain of restriction endonuclease FokI ( Table 2 ), and their engineering is also difficult. ZFNs and TALENs use engineered proteins to target specific DNA sequences in the genome. In contrast, CRISPR/CAS uses a small segment of engineered guide RNA to drive the CAS effector to specific locations in the genome ( Table 2 ). ZFNs and TALENs have an off-target effect on unwanted genes [ 54 ]. CRISPR-Cas is effective when provided with the correct template and relies on the development of specialized gRNA, which is easier to manufacture in comparison to the bulky protein guides required by ZFN and TALEN tools [ 55 , 56 ]. Table 2 compares and describes different features of CRISPR/Cas, ZFN, and TALEN systems. Additionally, under the right conditions, CRISPR-Cas can edit across plant, animal, and microbial genomes, posing advancements in genetic engineering across all living cells [ 56 , 57 ]. The CRISPR-Cas genome editing system was first used on animal cells in 2013 and poses biological risks and ethical concerns, as well as benefits [ 58 ]. CRISPR edits to the genome are irreversible and occasionally off-target. Mitigations to off-target effects rely on gRNA sequences that are complementary to possible off-target sequences. An additional issue with CRISPR-Cas editing falls on Cas nuclease binding and cleaving activity, as these processes are not consistently mutually exclusive. Mutations in the Cas9 protein can cause binding to occur without cleavage of target DNA [ 26 ]. Further research on the CRISPR-Cas delivery system is required to gain a full and complete understanding of its complications and potential side effects in order to expand on the role that CRISPR technology could play in genetic-based intervention therapies.

CRISPR genome editing technology has had a significant impact on various industries, including the biomedical, biotechnology, nanotechnology, plant, and livestock industries. One of the CRISPR applications is genome editing in the livestock industry to generate disease-resistant animals, improve animal welfare, and enhance productive traits [ 61 ]. Large animals, such as buffalo and cattle, have been the focus of this research. Moreover, CRISPR has proven to be useful in improving crops by enhancing their nutrient values [ 57 , 62 ]. CRISPR-based genome editing can potentially improve heart disease [ 63 ], neurological diseases [ 64 ], disease diagnosis [ 65 ], antiviral therapy [ 66 ], antibacterial disease [ 67 ], drug resistance [ 68 ], genetic disease [ 69 ], metabolic disease [ 70 ], biofuels [ 71 ], and many others. This technology holds great promise, and various industries are exploring its potential applications in different fields ( Figure 2 ).

The CAS system has been used in agriculture to modify grain shape, size, and weight [ 72 , 73 ]. It has also been used to improve the size and shape of tomatoes and fruits for better consumption and nutrient values [ 73 , 74 ]. Moreover, engineering through the CAS system changed the color of fruits and flowers [ 75 , 76 ]. Furthermore, the CAS system increased the shelf life of crops, such as tomatoes and bananas [ 77 , 78 , 79 ]. The shelf life of bananas is affected by ethylene during the preservation process. Genetic engineering of MA-ACO-1 through the CAS system delayed fruit ripening by two days and increased the shelf life of bananas [ 80 ]. Humans cannot synthesize carotenoids in their bodies, and diet is the only source. Carotenoids are important for eye disease prevention and antioxidant processes.

Interestingly, CRISPR/CAS was used to insert CrtI and PSY genes into rice, increasing the β-carotene content to 7.9 μg/g in dry weight [ 81 ]. Another example of crop improvement through the CAS system is increasing the γ-aminobutyric acid (GABA) content in food. GABA is a neurotransmitter inhibitor and functions as a regulator of anxiety and blood pressure [ 82 ]. Researchers have deleted SIGABA-Ts and SISSADH genes through the CAS system, which increased GABA levels by ~20-fold in tomatoes [ 83 ]. There are many other examples of crop improvement through the CAS system, suggesting a wide application of the CAS system in agriculture ( Figure 2 ). The CAS system has tremendous applications in livestock animals, improving the traits of large animals [ 84 ]. The CAS system has been used to produce disease-resistant animals [ 85 , 86 ], and it is also being used to control pests and insects [ 87 , 88 ].

4. Delivery Methods for CRISPR

The efficacy of CRISPR-Cas genome editing relies heavily on safe and predictable delivery systems in “in-vivo” (animals) [ 33 , 89 ]. Several delivery modes have been devised for integrating class II CRISPR-Cas-associated machinery into patients for therapeutic genome alteration ( Figure 3 ). These delivery systems include physical, viral, and non-viral vectors, posing mechanism-specific benefits and potential side effects to CRISPR-Cas and recipient health [ 89 , 90 ]. Many of the viral vector delivery methods for CRISPR/Cas are pathogenic and invoke immune responses in the presence of the virus [ 89 ]. Heightened immune responses make subsequent deliveries difficult and less therapeutic to the patient [ 89 , 91 ]. Immunogenicity can be an additional drawback to viral-based delivery systems when a patient has previously been infected with a respective virus [ 91 ]. Non-viral delivery modes have been explored to combat immunogenicity and insertional mutagenesis issues accompanying viral-based delivery systems [ 89 , 92 ]. Non-viral delivery of CRISPR/Cas has been utilized in cancer treatments due to low pathogenicity and high biocompatibility with patient tissues [ 89 , 92 ]. The viral vs. non-viral delivery route depends on the clinical application (in vivo, in vitro), cargo size, and potential pre-existing patient immunity to viral vectors [ 89 ]. Despite potential side effects, viral vector delivery methods in gene therapy are favored due to a virus’ capability of crossing cellular boundaries and storing extracellular cargo [ 89 , 93 ].

Adeno-associated virus (AAV)-mediated CRISPR/Cas delivery has many benefits, including low pathogenicity, long-term gene expression, and both in vivo and in vitro applications [ 89 , 94 ]. AAVs have an icosahedral capsid, a 26 nm diameter, and a single-stranded genome with ~4.7 kb [ 95 ]. The AAV family is defined by its expansive serotype diversity and tropism, making it an attractive vehicle for gene therapy [ 89 , 96 ]. More than 3000 clinical trials that use AAV as a gene delivery vector are ongoing [ 97 , 98 ]. For example, a few gene therapies based on AAV vectors (Zolgensma, Luxturna, and Glybera) have been approved for human use [ 99 ]. AAV-mediated CRISPR delivery has been effective for both neural gene therapy and Duchenne muscular dystrophy (DMD) treatment in mice [ 100 , 101 ]. The AAV-mediated delivery of Cas9 and sgRNA to mouse models affected by a nonsense point mutation in the dystrophin gene (exon 23) successfully deleted the exon and subsequently decreased the DMD phenotype [ 102 ]. The CRISPR-associated protein SpCas9 is widely used for gene editing purposes. However, its size is a major limitation in packaging into a single AAV vector (packaging capacity is ~4.7 kb, including ITRs) [ 89 , 103 ]. As a result, the protein and sgRNA must be encoded on different vectors, which can pose a significant challenge for manufacturers [ 89 , 101 , 104 ]. However, alternative Cas proteins such as SaCas9, CjCas9, Neisseria meningitides Cas9 (NmCas9), Streptococcus thermophilus Cas9 (St1Cas9), and others are smaller in size and can be packaged into a single AAV, making gene therapy easier and more efficient [ 105 , 106 , 107 ]. Additionally, the discovery of compact Cas12f has opened many opportunities for scientists to use AAV as a delivery vector for various gene therapy and genome editing [ 108 ]. This is discussed in more detail in the miniature Cas section.

An alternative viral vector for the delivery of CRISPR-Cas is the retrovirus family, known as lentivirus (LV) ( Figure 3 ), which has a single-stranded genome of 7–12 kb [ 109 ]. Similar to AAVs, LVs provide effective cellular transduction across various cell types (in vivo and in vitro), low immunogenicity, and safe delivery of gene editing machinery [ 89 , 110 ]. LVs for gene therapy were originally derived from the human immunodeficiency virus (HIV-1) and have since been altered for cell-specific transduction [ 111 ]. Despite being relatively easy to design, LVs typically integrate their genome into the host genome, subsequently elongating the process of gene expression [ 112 ]. The manipulation of LV via an integrase mutation has led to the development of “integration-deficient” lentiviral vectors, reducing the risk of continuous Cas overexpression and off-target effects [ 113 ]. Notably, the RNP (ribonucleoprotein) contains CRISPR-Cas and sgRNA and can be delivered by lentivirus or lentivirus-like particles without genome incorporation, thus eliminating the risk of off-target effects [ 114 ]. LV-associated delivery has been successful in preclinical trials for hemophilia gene therapy and Gaucher Disease [ 89 , 115 , 116 ]. The ex vivo LV-mediated delivery of CRISPR/Cas to hematopoietic and pluripotent stem cells has shown promise in phase I and II clinical trials [ 89 ]. The European Medicines Agency and the Food and Drug Administration (FDA) have approved LV for use [ 89 ]. This means that LV has met the safety and efficacy standards required by these regulatory bodies and is now authorized for medical treatments.

The non-viral vectors for the delivery of CRISPR-Cas have demonstrated several benefits, including minimal cost for wide-scale production, high biocompatibility, and low pathogenicity [ 117 ]. Lipid nanoparticles (LNPs) are amphiphilic molecules composed of ionized or cationic lipids engineered to deliver exogenous materials to cells, including nucleic acids and CRISPR-Cas machinery ( Figure 3 ) [ 117 ]. Compared to other viral and non-viral delivery methods for gene therapy, LNPs pose advantages that make them attractive vehicles for administering cancer drugs, such as lowering drug toxicity and preventing drug degradation [ 118 ]. Cas and sgRNA mRNA, as well as the Cas:sgRNA RNP complex, have been correctly loaded onto LNPs and efficiently transported to the livers of mice for gene alteration [ 119 ]. The delivery of the CRISPR-Cas system via LNPs to tissues outside of the liver has yet to be efficiently explored and remains an issue when utilizing LNP delivery in preclinical settings [ 89 ]. To combat the COVID-19 pandemic, a highly successful mRNA vaccine was created to limit COVID-19 infection. Similar vaccine technology can be utilized to transmit CRISPR/Cas via LNPs, improving the efficacy of the solid nanoparticle delivery methods.

5. Miniature Cas12f

Neither SpCas9 (1368 amino acids) nor AsCas12a (1307 amino acids) can be packaged into a single AAV vector for efficient in vivo delivery. Although smaller Cas9, such as saCas9, cjCas9, nmCas9, etc., have been identified and used in all-in-one AAV vectors, packaging and transduction efficiency remain to be optimized [ 105 , 106 , 107 ]. Recently, researchers have identified super compact CRISPR/Cas effectors, such as Cas12f and Casϕ, which are classified as a type V-F system. These Cas effectors are 400–700 amino acids long and have shown genome editing activity in human cells [ 12 , 120 , 121 ]. The development of smaller CRISPR effectors and miniCAS shows great promise in overcoming the limitations of AAV and expanding the range of potential gene editing targets.

The first study identified Cas14 from an uncultured archaeon (Un1), initially named Cas14a1 and later renamed Un1Cas12f1, and evaluated the targeting specificity and activity using a single-stranded DNA in a PAM-independent manner but not in mammalian cells [ 17 ]. Several later studies engineered the protein and/or sgRNA scaffold of the Un1Cas12f1 system and demonstrated high editing efficiency on double-stranded DNA in a PAM-dependent manner and mammalian cells [ 18 , 122 , 123 ]. Un1Cas12f1 is also active for base editing [ 124 ] and gene regulation [ 125 ].

A recent study has identified a smaller Cas 12f version from Acidibacillus sulfuroxidans called AsCas12f ( Figure 4 ). This version contains only 422 amino acids, has a PAM with TTR (where R is G or A), and has shown comparable genome editing efficiency in human cells [ 122 , 126 ]. This discovery is a game changer, opening exciting new opportunities for scientists to utilize the mini AsCas12f for genome editing in humans, with potential applications in gene therapies using AAV delivery vectors. Figure 4 illustrates the comparison of the length and size of three representative Cas enzymes—SpCas9, FnCas12a, and AsCas12f.

Recent studies on sgRNA engineering [ 122 , 124 ] and the structure of the Cas12f-sgRNA complex [ 127 , 128 , 129 ] have brought groundbreaking developments in genome editing research. Using cryoEM, researchers have uncovered the structure of AsCas12f, which forms a dimer structure similar to UnCas12f [ 108 ]. High-throughput screening using deep mutational sequencing (DMS) and structural design information has identified two AsCas12f variants, AsCas12f-HKRA and AsCas12f-YHAM, which have significantly enhanced genome editing activity of AsCas12f, called enAsCas12f, in mammalian cells, comparable to SpCas9 and AsCas12a, which is significant progress in the field [ 108 ]. Moreover, researchers have engineered an optimized sequence for sgRNA by deleting certain stem loops, which has led to better efficacy in human cells. To prove the application of enAsCas12f, the authors used an AAV vector to deliver enAsCas12f in human cells and determine the efficacy against six internal loci, such as VGFA, HEXA, PDCD1, TP53, and APOB, and found comparable editing to AsCas12a. EnAsCas12f also has comparable editing efficiency to SpCas9 and AsCas12a against the most promising therapeutic targets, such as PCSK9, ANGPTL3, and TTR. Furthermore, the researchers have demonstrated the efficacy of enAsCas12f in vivo using a mouse model.

The authors constructed a hepatotropic AAV serotype (AAV8) by inserting the AsCas12f or AsCas12f-HKRA variant under the HCRhAAT promoter and sgRNA (under U6 promoter) specific for the TTR gene (transthyretin amyloidosis). They then evaluated its therapeutic potential in 7-week-old mice by determining the expression of transthyretin in the plasma of the treated mice. Impressively, they found that the AsCas12-HKRA variant was able to dose–dependently reduce the level of transthyretin, with a high editing rate of approximately 66.3% achieved at the target location after only eight weeks of AAV-based gene delivery in the injected mice [ 108 ]. Next, the authors used the AsCas12f-HKRA variant to insert the EGFP gene at the Alb locus and found that there was a significant increase in the level of EGFP expression in the injected mice. They then applied a similar strategy to knockin coagulation factor IX at the Alb locus in hemophilia mice. They found that enAsCas12f increased the expression of factor IX in the plasma of treated mice [ 108 ]. Most significantly, they demonstrated high efficiency of factor IX positional knockin in mice using an all-in-one AAV vector encoding enAsCas12f and sgRNA and carrying a donor sequence (factor IX with both homology arms) with a self-cleaving sgRNA target site, which can eliminate the AAV vector after gene editing and knockin [ 108 ]. Finally, the scientists validated the efficacy of enAsCas12f in epigenome editing in mouse studies using an all-in-one AAV vector encoding denAsCas12f-VP64-PT2-MS2-P65-HSF1 and sgRNA in either universal CMV or liver-specific TTR promoters [ 108 ]. These groundbreaking developments in the Cas12f-sgRNA complex have the potential to revolutionize genome editing, paving the way for new possibilities in future research and therapeutic applications ( Figure 4 ). The results of these studies are highly promising and will undoubtedly shape the future of genome editing research and particularly clinical trials.

6. Biomedical Applications of CRISPR/CAS

The CRISPR-CAS system has wide applications in biomedical sciences. The CAS system has been used to generate robust and reproducible model systems such as transgenic animals, brain organoids, IPS-derived cells, and cancer models to study various diseases, which was challenging before. For example, the CAS system produced a leukemia disease model by knocking out multiple genes simultaneously [ 130 ]. Indeed, the CAS system was used to generate iPSC-derived neurons that display characteristics and phenotypes of Alzheimer’s disease [ 131 ]. Creating an accurate disease model for Alzheimer’s disease is challenging. However, by leveraging the CAS system, it was possible to generate an accurate disease model for Alzheimer’s disease. Another biomedical application of the CAS system is high-throughput screening to better understand the cellular pathways and molecular mechanisms and identify genes involved in a disease [ 132 , 133 ]. The CAS system has been used to accurately diagnose diseases in a timely manner; this is very important for disease treatment and epidemiological monitoring of diseases. CRISPR-based diagnoses are accurate, fast, affordable, and can be performed at home. For example, CRISPR/CAS-based diagnosis methods, such as NASBACC (nucleic acid sequence-based amplification CRISPR cleavage), are ultrasensitive methods to detect Zika virus in the femtomolar range [ 134 ]. The CAS system was used to develop SHERLOCK diagnostic tools (specific high-sensitivity enzymatic reporter unlocking). SHERLOCK is currently being used to detect single DNA or RNA molecules in samples from dengue, Zika, and pneumonia patients [ 135 , 136 ]. SHERLOCKv2 is a highly sensitive method that detects target sequences at the femtomolar range (10 −21 ). Moreover, the CAS system was utilized to develop DETECTOR (DNA endonuclease-targeted CRISPR trans reporter) diagnosis tools to discriminate between HPV16 and HPV 18 more precisely [ 137 ]. In biomedicine, the CAS system has been employed in treating hereditary diseases such as hemophilia [ 138 , 139 ], β-thalassemia [ 140 , 141 ], cystic fibrosis [ 142 , 143 ], Alzheimer’s disease [ 144 ], Huntington’s disease [ 145 ], Parkinson’s disease [ 146 ], Tyrosinemia [ 147 ], Duchenne muscular dystrophy [ 148 ], Tay–Sachs disease [ 149 ], Fragile X syndrome [ 150 ], phenylketonuria [ 150 ], blindness [ 151 ], cataracts [ 152 ], cancer [ 153 ], blood disorders [ 154 ], and others. The CRISPR system has been used to study and treat infectious diseases in humans [ 155 ]. There are many other biomedical applications of the CRISPR/CAS system. The CRISPR/CAS system has revolutionized the field of gene therapy. Figure 2 describes the various applications of the CRISPR/CAS system.

Recently, CRISPR-based technology developed STOPCovid.v2 to detect SARS-CoV-2 in infected patients. This detection method achieved a specificity of 98.5% and a sensitivity of 93.1%, which is higher than RT-PCR for SARS-CoV-2 detection. STOPCovid.v2 was approved by the FDA for SARS-CoV-2 detection [ 156 , 157 ].

7. CRISPR/Cas-Based Gene Therapies and Therapeutic Applications

The first CRISPR-based gene therapy in humans provided evidence for its application to treat both genetic diseases and infectious diseases [ 158 ]. About 6000 diseases are caused by genetic disorders, and the majority of them do not have effective treatment; thus, CRISPR provides an excellent opportunity to treat these genetic diseases. In 2020, Jennifer Doudna (University of California, USA) and Emanuelle Charpentier (Max Planck Unit for the Science of Pathogens, Germany) were awarded the Nobel Prize in chemistry for their work on endonuclease, the CRISPR-CAS system for gene editing. Gene therapy requires the replacement of a defective gene with a functional gene or the correction of mutations in a gene at its native location. A list of CRISPR-Cas systems that have been used in genome editing is described in Table 3 . The Cas effector protein used, its classification based on the type and organism of origin, and the protospacer-adjacent motif (PAM) sequence that it recognizes are summarized in Table 3 . In the SpCas9 system, sgRNA prefers the NGG PAM sequence, which is present in most organisms, thus facilitating genome editing in animals and plants [ 41 , 159 ]. The SpCas9 effector has been applied to many diseases, including cancer, sickle cell anemia, cardiovascular diseases, and neurodegenerative diseases [ 160 , 161 , 162 , 163 ]. Interestingly, SpCas9 was modified by mutating its structural domain to generate a dead Cas9 (dCas9) and fused it with other functional proteins such as an adenine base editor, a cytosine base editor, and prime editors to correct deleterious point mutations specifically. Similarly, other Cas effectors were identified and characterized as described in Table 3 . Another example of CAS effectors includes Cas13a, which was isolated from Leptotrichia shahii and shown to have cleavage functions in many studies [ 41 , 164 ]. Compact Cas12f was also identified as having comparable genome editing to SpCas9 [ 108 ]. Notably, specific Cas9 variants have desirable properties that are utilized in various circumstances. For example, amongst the Cas9 proteins present in mammalian cells, XCas9 has a high PAM compatibility that helps with numerous processes within human cells [ 165 ]. Thus, the high-fidelity Cas9 variant is able to recognize stricter PAM sequences, which reduces the risk of unintended DNA cleavage from happening. Table 3 also mentions unique Cas effectors, such as Cas12a and Cas13. Cas12a is able to target single-stranded DNA specifically [ 122 ]. Cas13 targets RNA, which makes it valuable for cleaving RNA viruses, like HIV or influenza [ 166 ]. This diverse range of Cas effector proteins shown in Table 3 offers many advantages for CRISPR technology. Since different Cas effector proteins, such as Cas9, Cas12a, and Cas13, recognize and cleave distinct types of genetic material, researchers can choose from many options to select the most appropriate tool.

7.1. CRISPR Technology and Cancer

It is well known that each cancer patient has unique genetic and epigenetic variations that result in different reactions to the same treatment. CRISPR-Cas-based techniques have become a pivotal tool for advancing personalized treatment options in the context of cancer. CRISPR-Cas methods allow researchers to identify specific genetic factors influencing cancer development and formulate personalized therapeutics tailored to the individual [ 187 ]. CRISPR/Cas provided a powerful example of its therapeutic capacities by pinpointing and targeting the KRAS oncogene, subsequently preventing cancerous growth [ 188 ]. Before the advancement of CRISPR technology, gene editing tools, such as ZFNs and TALENs, were utilized, but they had many limitations [ 189 ].

Genome-wide CRISPR screening is a powerful tool for understanding and exploring the intricate contributions of diverse genetic elements to the complex landscape of cancer progression. The backbone of this method incorporated the usage of gRNA libraries, containing guide RNAs that are tailored for specific genetic elements [ 187 ]. CRISPR screens are deployed to navigate the genetics of different types of cancers by revealing regulators of genetic dependencies, synthetic lethal gene interactions, and drug targets across various malignancies [ 187 ]. Studies being executed on pulmonary cancer treatment state the prospects of CRISPR-CAS single handedly becoming the instrument for treating this disease by editing genes [ 189 ].

There are a few methods of the effective delivery of CRISPR-Cas gene editing for cancer therapy. Electroporation is a commonly used physical method that delivers CRISPR-Cas into tumor cells in vitro. Its main feature temporarily disrupts lipid bilayers, enhancing cell membrane permeability [ 188 ]. Viral vectors are another delivery mechanism but pose challenges for packaging large genes [ 188 ]. Non-viral vectors, such as commercial transfection reagents, have demonstrated promise in delivering CRISPR-Cas systems in vitro [ 188 ]. The ex-vivo delivery of CRISPR-Cas tools involves treating cells outside the body prior to implementation into the body [ 188 ]. In chimeric antigen receptor (CAR) T-cell therapy, a patient’s T-cells are genetically engineered to express receptors that recognize and bind to cancer cells. CAR T-cell therapy has shown success in treating various types of cancer, particularly B-cell malignancies [ 190 , 191 ]. The US FDA has approved CAR T-cell therapies for the treatment of B-cell lymphoma [ 192 ]. However, the use of this therapy on other types of cancer, such as solid tumors, is not as effective [ 193 , 194 , 195 ]. There are some challenges associated with the CAR T-cell approach, including cytokine-related toxicities, limited CAR T-cell expansion and persistence, and T-cell exhaustion. CAR T-cell hypofunction [ 196 , 197 ], limited CAR T-cell expansion and persistence, and premature senescence [ 196 ] are significant reasons for CAR T-cell therapy failure. CRISPR-Cas has been used to enhance T-cell persistence and effector function by removing negative regulators through gene editing [ 198 , 199 , 200 ]. The CRISPR-Cas system can also be used to precisely engineer CAR T-cells [ 201 ]. Studies have shown that targeting diacylglycerol kinase (DGK) using the CRISPR-Cas system can make CAR T-cells more resistant to immunosuppression [ 202 ]. Additionally, deleting genes responsible for cytokine release syndrome and neurotoxicity, such as IL-6 and GM-CSF, can improve CAR T-cell efficiency [ 203 , 204 ]. Interestingly, the CRISPR-Cas system has also been used to insert CAR cassettes at specific locations in the genome [ 205 ]. The diverse array of CRISPR-Cas delivery systems has underscored the ongoing exploration of innovative approaches to cancer gene therapy.

7.1.1. Lung Cancer

Lung cancer is a complex disease that manifests in two primary forms: small-cell lung cancer and non-small-cell lung cancer [ 206 ]. Lung cancer has been identified as the second most diagnosed type of tumor, causing 1.8 million cancer-induced deaths [ 207 ]. Since cancer drug resistance has been the main culprit in providing relief to lung cancer patients, CRISPR techniques have offered a glimpse of hope for a better quality of life [ 206 ].

CRISPR-Cas systems are being utilized to edit oncogenic gene mutations, like EGFR [ 208 ]. EGFR is a tyrosine kinase, and mutations in its kinase domain are found in approximately 10% to 40% of non-small lung cancer patients. These mutations are mainly present in exon 19 and exon 21 of the EGFR gene [ 209 , 210 ]. A study has demonstrated that a CRISPR-Cas system and a donor template with homology arms can be used to replace mutated EGFR with normal or wild-type EGFR [ 211 ]. Moreover, the CRISPR-mediated knockout of mutated EGFR can prevent cancer cell growth and proliferation [ 206 ]. This gene elimination stops rapid cell growth and cell survival in vitro and in vivo [ 208 ]. CRISPR can also strategically insert genes that enhance cancer cell drug sensitivity [ 206 ]. Editing genes with cancer cells gives the opportunity to trigger apoptosis or even make the oncogenes vulnerable to alternative cancer therapies [ 208 ]. This approach effectively targets cancer cells and minimizes harm to healthy tissues. Researchers administered Adv-Cas9-sgG12S to A549 mice, resulting in a significant 30% reduction in tumor volume. The study also found that the expression of the mutant KRAS protein was extensively reduced, offering hope for those suffering from this devastating disease [ 212 ]. The use of a light-inducible CRISPR-Cas system to cleave the mutated BRAF gene is another significant development in cancer treatment. This approach induced melanoma cell apoptosis, leading to the inhibition of cell proliferation, migration, and invasion [ 213 ]. Patients with non-small lung cancer with CRISPR gene editing performed on their T-cells were observed. The off-target activity was only 0.05%, and edited T-cells showcased no adverse effects [ 208 ]. Compared to chemotherapy, CRISPR has been deemed more successful due to its ability to target epigenetic modifications in cancer and reverse the changes that make cancer cells proliferate and divide uncontrollably [ 214 ].

The possibilities of CRISPR have been marked beyond its therapeutic potential. With this, blood tests can utilize CRISPR to identify cancer cells’ gene signatures [ 214 ]. This personalized approach can help identify the disease at early stages when the most effective treatment can be administered [ 214 ]. The current perspective on CRISPR suggests that gene editing will be more efficient with continuous advancements in delivery methods that are employed in vivo [ 207 ]. CRISPR is gradually becoming a reliable tool for lung cancer due to its high efficacy in genome editing and minimal off-target activity [ 214 ].

7.1.2. Brain Cancer

Glioblastoma multiforme (GBM) is a brain cancer that has been challenging to treat and has a poor prognosis. The emergence of CRISPR-Cas gene editing has led to recent advancements and promising brain cancer research and therapy. Some of the CRISPR-Cas-based clinical trials are listed in Table 4 .

CRISPR methods are now making promising strides in silencing oncogenes. Researchers have utilized CRISPR technology to eliminate a specific gene in various cancer cell lines and monitor its impact on the growth of cancer cells. As a result, a DepMap database of cancer-related genes comprising data from thousands of cancer cell lines from numerous tumor types has been established [ 215 ]. CRISPR can potentially disrupt genes encoding growth factors, like EGFR and PDGFR [ 216 ]. By doing so, researchers can inhibit their pro-tumorigenic signaling pathways in an effort to starve the cancer cells of the growth signals they require to proliferate [ 216 ]. A study has employed a whole-genome CRISPR screen to identify the genes that are accountable for irradiation resistance in GBM cells. The authors of this study have used a human CRISPR activation library, which includes approximately 70,290 sgRNA that specifically targets 23,430 genes. The genes that are responsible for irradiation resistance in GBM cells were identified [ 217 ]. CRISPR is being used to edit genes encoding transcription factors that control gene expression. Specifically, MYC is an essential transcription factor that drives glioblastoma growth and survival [ 218 ]. CRISPR can target MYC and silence the gene in order to suppress further tumor progression [ 218 ]. CRISPR is being employed to insert genes encoding apoptotic proteins that are key for triggering cell death [ 219 ]. CRISPR can deliver the apoptotic gene p53 that eventually causes tumor cell death in glioblastoma models [ 219 ]. Utilizing this method directly eradicates cancer cells from the tumor mass. It is apparent that glioblastoma cells develop resistance to conventional therapies [ 220 ]. This occurrence is due to their robust DNA repair mechanisms [ 220 ]. CRISPR can overcome this complex design and disrupt genes involved in DNA repair [ 220 ]. CRISPR tools are used to edit immune cells with the help of chimeric antigen receptors [ 221 ]. These receptors are able to recognize and attack specific tumor antigens, which is essential for CAR T-cell therapy for glioblastoma treatment [ 221 ]. CRISPR offers a personalized approach for targeted therapies based on the patient’s tumor and unique genetic makeup [ 219 ]. The versatility of CRISPR gene editing makes it a promising method for halting the growth and proliferation of glioblastoma oncogenes [ 221 ].

7.2. Sickle Cell Anemia and Beta-Thalassemia

Sickle cell anemia is a genetic blood disorder that stems from abnormal hemoglobin, which is the culprit for health complications in the patient. Beta-thalassemia is another inherited blood disorder that disrupts beta globin production in the body. The emergence of CRISPR-based techniques has offered a new and advanced method to tackle these diseases.

Researchers have utilized CRISPR-Cas to pinpoint the BCL11A gene in stem cell patients with beta-thalassemia [ 222 ]. The gene editing in this clinical trial has allowed fetal hemoglobin production that makes reparations for the defective adult hemoglobin in the patient [ 222 ]. This system works by initiating a CRISPR screening to detect the regions of the HBG1 and HBG2 promoters [ 223 ]. These promoters are where the sgRNA sequences’ target sites are present, which serves as a guide for the Cas protein to locate and target the BCL11A binding sites [ 224 ]. CRISPR gene editing allows the repressor proteins (BCL11A and LRF) to switch on the production of fetal globin by preventing them from binding to the HBG1 and HBG2 promoters. This CRISPR technology has significantly improved red blood cell functions and patients’ overall well-being with transfusion-dependent B-thalassemia or sickle cell disease [ 222 ].

The challenges of using CRISPR include off-target editing and risks associated with off-target mutations [ 225 ]. CTX001, a form of CRISPR gene editing therapy, was observed to have high edited allele frequencies of 68.9% [ 222 ]. CRISPR techniques can pose issues with off-target mutations, severely disrupting normal gene functions [ 224 ]. Due to the long-term nature of CRISPR editing, the off-target activity could build up and showcase side effects in the patient’s body [ 224 ]. This issue can be overcome using rational sg RNA sequences and a more efficient and specific Cas protein [ 224 ]. Lack of knowledge surrounding the long-term side effects of CRISPR gene therapy necessitates careful and prolonged monitoring of patients. Due to these extensive hospitalizations, readmissions, and check-ups, many patients and family members have reported the financial strain that this therapy has caused them [ 225 ]. Additionally, it is crucial to note that gene editing success rates depend on the patient’s phenotype. Therefore, the results are different among each individual who receives CRISPR-based gene therapy [ 226 ].

7.3. Cystic Fibrosis

Cystic fibrosis (CF) is a genetic disorder that affects the lungs, pancreas, and other organs. The leading cause of CF is mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. The emergence of CRISPR-Cas gene editing has alleviated some of the challenges endured by CF patients through therapeutic interventions [ 227 ].

CRISPR-Cas gene editing methods can be used to correct the mutated CFTR gene in the patient [ 227 ]. It is understood that this strategy can directly edit CFTR genes in the airway epithelial cells in order to restore the gene’s function [ 100 ]. CF patients also experience chronic inflammation and progressive lung damage. CRISPR techniques can be utilized to edit genes involved in inflammation pathways [ 228 ]. More specifically, this technology could complement existing iPSC-based approaches, which target lung epithelial cells relevant to CF lung disease [ 18 ]. Overall, this treatment helps to reduce lung damage and improves respiratory function in the CF patient.

Researchers are using the CRISPR-Cas technology and applying it to animal models, like knockout sheep, rats, and mice [ 227 ]. Animal models provide a platform for testing potential treatments and drugs before moving to human trials. CRISPR-generated CF models can be used to test gene editing therapies to correct specific mutations [ 227 ]. CRISPR’s versatility extends beyond directly targeting the CFTR gene. CRISPR can be used to edit genes involved in protein folding and trafficking [ 18 ]. This new avenue allows researchers to improve protein function, even when CFTR mutations are present [ 18 ]. CRISPR offers a promising approach for treating CF because it can be adapted to target a variety of CF mutations and personalize treatment based on the patient’s genetic makeup [ 18 ]. Prime editing makes this possible, where researchers are able to edit a specific DNA sequence of different lengths [ 229 ]. Since CFTR genes are large, prime editing can repair the disease-causing variants in an efficient manner [ 229 ].

7.4. Multiple-Sclerosis

Multiple Sclerosis (MS) is a chronic autoimmune disease that aggressively attacks an individual’s nervous system. This disease leads to progressive neurological deterioration. Emerging gene editing technologies, like CRISPR-Cas, offer relief for MS patients.

CRISPR methods are being used to edit genes in immune cells [ 230 ]. This gene editing inhibits healthy myelin from being attacked [ 230 ]. CRISPR techniques can be used to edit genes that are involved in myelin production and repair within oligodendrocytes [ 231 ]. Oligodendrocytes are the cells that are responsible for myelin formation [ 231 ]. This approach aims to stimulate the body to repair the damaged myelin naturally and halt the progression of the disease [ 231 ]. The candidate genes for CRISPR-Cas editing are IL7R, DDX39B, IL2RA, and TNFRSF1A [ 230 ]. Targeting these specific genes in immune cells makes it possible to reduce the overactive immune response that is a response to MS progression [ 230 ]. CRISPR serves as a major tool for research. Researchers are using CRISPR methods to create more precise models of MS by introducing mutations into the genomes of mice [ 232 ]. This approach allows researchers to study the models that closely resemble human MS and test potential therapies in a more relevant setting [ 232 ]. Autoreactive T-cells are the contributors to tissue injury in individuals with MS. Studying CRISPR methods now explains the immunological processes that dictate autoreactive T-cells [ 232 ]. Researchers can also use CRISPR methods to systematically disrupt genes in immune and nervous system cells [ 232 ]. This way, they can identify new genes that are involved in MS pathogenesis. An important feature of CRISPR is the ability to have personalized treatment strategies. CRISPR-based tools help identify genetic variations that contribute to a patient’s specific MS case [ 232 ].

Utilizing CRISPR methods requires careful evaluation and consideration of the potential risks and long-term repercussions [ 230 ]. Overall, MS development can be prevented and mitigated by correcting disease-associated mutations in specific genes.

7.5. HIV Gene Therapy

Human immunodeficiency virus, also known as HIV/AIDS, poses a significant health challenge to the human population globally. It is important to note that 38 million people are infected with HIV, and 800,000 people lose their lives due to HIV-related infections every year [ 233 ]. This virus attacks the body’s immune system and affects the quality of life of infected patients. No vaccine is available to cure HIV. The emergence of CRISPR-Cas gene editing techniques has allowed for better treatment options for people fighting against HIV.

CRISPR/Cas genome editing has been extensively employed to fight various types of viruses, including HIV [ 233 , 234 , 235 , 236 , 237 ]. Table 5 comprehensively summarizes various CRISPR-Cas-based gene therapies against HIV that target both viral and host factors. The studies have demonstrated significant progress in using the CRISPR-Cas system to remove proviral DNA from infected cells, which is a significant milestone in the fight against HIV [ 165 , 235 , 236 , 237 , 238 ]. The research has shown that focusing on highly conserved viral genes, such as the Rev gene, is a promising strategy, as it can eliminate the risk of virus escape and provide a more effective solution against HIV [ 239 , 240 , 241 ]. The stable transfection of cultured cells with plasmids containing spCas9 and duplex sgRNAs has shown great potential in excising proviral DNA from myeloid cells [ 128 , 235 ]. Moreover, the success of saCRISPR in eliminating HIV from infected cells by targeting multiple components of its proviral DNA, including 5′ LTR, 3′ LTR, and gag-pol, is a remarkable breakthrough in this field [ 238 ]. Lentiviral gene delivery of spCas9/sgRNA targeting the LTR U3 region to HIV-infected primary CD4 + T-cells from healthy human subjects and HIV patients [ 234 ] also induces effective HIV eradication. A combination of sgRNAs targeting the LTRs and the viral structural genes provides a more efficient means for HIV eradication in cultured cells [ 242 ]. The identification of a smaller saCas9 renders a rapid and extensive application of AAV in animal gene knockout [ 106 , 243 , 244 ] and knockin [ 245 , 246 ] studies. AAV9-mediated saCas9/duplex sgRNA can excise the integrated HIV genome in HIV-transgenic mice and rats [ 247 ]. AAV-DJ8-mediated quadruplex sgRNAs/saCas9 can excise HIV proviral DNA in several tissues and organs of HIV-infected hu-BLT mice [ 238 ]. Recently, the AAV9-saCas9/duplex sgRNA system was used to excise the SIV proviral genome in different organs/tissues, including the brain [ 248 ]. Studies have shown that RNA-targeted gene editing can eliminate latent HIV infection and prevent new virus production [ 235 , 242 ]. These studies clearly demonstrated the potential of CRISPR and its breakthrough application in disease treatment for infectious diseases. CRISPR-Cas can introduce fragmental deletion of proviral DNA in the infected cells [ 249 ]. This allows for the prevention of viral replication, which would help achieve a permanent cure for the virus [ 233 ]. CRISPR methods can also boost the immune system by editing the genome of immune cells, like T-cells [ 250 ]. Incorporating chimeric antigen receptors (CARs) allows CRISPR to distinguish and ultimately attack the infected cells [ 250 ]. This combination of CRISPR and Car T-cell therapy can be utilized for HIV treatment. Additionally, CRISPR can be used to edit genes involved in immune recognition pathways. This allows the immune system to be more efficient and likely to both detect and eliminate the HIV-infected cells in the patient’s body [ 251 ]. This approach aims to ultimately strengthen the patient’s natural defense mechanism against the virus [ 251 ]. When CRISPR-Cas systems are introduced into humans, there is a risk of subsequent immunogenicity in the patient [ 233 ]. To combat this, researchers can use a non-immunogenic Cas system, immune-suppressive drugs, or deliver the Cas proteins as mRNA [ 233 ]. Hence, combining different CRISPR-Cas systems to target the multifaceted HIV will be more effective if designed carefully. HIV may develop resistance against current drugs. CRISPR methods are employed to target the mutations that cause this level of resistance [ 252 ]. CRISPR is a personalized treatment method because it can strategically analyze individual patient genomes to identify specific viral mutations or the immune system’s weakness [ 250 ] and design personalized sgRNA for HIV proviral eradication. This way, patients can receive a specialized treatment option to fight against the virus present in their bodies. The development of 3D microglia-containing brain organoids to study HIV pathogenesis and CRISPR application in HIV eradication has been developed [ 253 ]. These 3D brain organoids represent valuable resources for studying HIV infection and latency and explore the effective delivery of the CRISPR/Cas editor for HIV eradication.

The CRISPR system has been used in many clinical trials for genome editing. CRISPR was used to disrupt the CCR5 gene in hematopoietic stem cells and progenitor cells obtained from patients to treat HIV-1 infection [ 268 ]. In another clinical trial, CRISPR-edited HSPC cells were engrafted into a 27-year-old man with HIV-1 infection. The results demonstrated that CCR5 gene disruption efficacy was increased from 5.2 to 8.3% in cells over 19 months ( NCT03164135 ). Promising results were obtained from a recent clinical trial using CRISPR-based genome editing for sickle cell anemia [ 222 ]. In this study, CRISPR-based genome editing was performed in patient-derived HSPC cells targeting the BCL11A enhancer to produce an engineered HPSC called CTX001. Clinical trials were initiated to treat advanced and uncurable cancer, including multiple lymphoma and liposarcoma, by the CRISPR-based gene editing of patient-derived T-cells [ 269 ]. This study used CRISPR to suppress endogenous T-cell receptors (TCRs) and PD-1. Engineered T-cells were injected back into the patient, and three patients were treated using this method. A list of clinical trials that used CRISPR-based gene editing is described in Table 4 .

7.6. Liver Diseases

The liver is essential for body homeostasis, drug detoxification, proteins, and lipid metabolism. CRISPR-based gene therapies have been used to treat many diseases that affect the function of the liver in the body. The most crucial liver disease is phenylketonuria (PKU), caused by a mutation in PAH, which encodes for phenylalanine hydrolase (PAH). PKU is an autosomal recessive liver disorder in which the level of phenylalanine increases in the blood when PAH activity is reduced, leading to neurotoxic effects. In severe cases of PKU, a point mutation Arg408Trp in PAH is the most prevalent [ 270 ]. When PAH activity is reduced, the level of phenylalanine in the blood increases, leading to neurotoxic effects. A recent study used an rAAV vector and CRISPR with a cytidine base editor to correct PAH mutation. About 63% of the mRNA correction rate was achieved, and a normal phenylalanine level was restored post-gene therapy in these mice [ 271 ]. Treated mice showed reduced growth retardation after genome editing in the homozygous Pah enu2 mouse model. Wilson’s disease (WD) is a rare genetic disorder caused by a mutation in the ATP7B gene, a copper-transporting P-type ATPase. AAV-based gene therapy in the WD mouse model reduces the copper level in these mice after six months of administration of gene therapy [ 272 ]. In another study, hepatitis B virus (HBV)-infected cells were targeted by the CRISPR/CAS9 system using nanoparticles to inactive HBV covalently closed circular DNA [ 273 ]. CRISPR/CAS-based gene therapy has been employed to treat other liver diseases such as Tyrosinemia type I (HT1) [ 245 ], Argininemia [ 274 ], Alpha-1 antitrypsin deficiency (AATD) [ 275 ], hemophilia [ 276 ], and ornithine transcarbamylase (OTC) deficiency [ 104 , 277 ].

7.7. Cardiovascular Diseases

Cardiovascular diseases are responsible for death in humans globally [ 278 ]. Cardiovascular diseases that affect humans include heart attack, aortic dissection, atherosclerosis, and myocardial hypertrophy [ 279 , 280 , 281 ]. Direct CRISPR delivery to cardiomyocytes may lead to off-target effects. Using CRISPR technology, a dCAS9/VPR transgenic mouse was developed, where dCAS9/VPR was expressed using cardiomyocyte-specific promoter Myh6 [ 282 ]. The administration of AAV9 harboring sgRNA activates gene transcription that promotes cardiomyocyte proliferation. This study holds great promise because cardiomyocyte proliferation decreases with aging, which can be restored using CRISPR-based gene therapies. In another study by Zhang et al., CRISPR was specifically delivered to vascular smooth muscle cells using a hydroxyl-rich lipid nanoparticle [ 283 ]. A study has specifically delivered CRISPR to endothelial and VSMCs using lipid and polymer nanoparticles [ 284 ].

8. CRISPR and Drug Approaches

Medicine is experiencing a transformative shift as CRISPR-Cas gene editing technologies gain popularity. This powerful tool is being used alongside traditional drug development approaches. Combining these methods allows researchers to discover avenues for tackling several complex diseases.

It is vital to point out that CRISPR techniques enhance drug sensitivity. CRISPR edits genes involved in drug resistance pathways, which makes cancer cells more prone to traditional therapies [ 285 ]. A considerable example is the ability of CRISPR to enhance the effectiveness of chemotherapy drugs that are involved with leukemia treatment [ 285 ]. With this, patients’ bodies can overcome resistance, and the treatment’s outcomes are greatly improved. CRISPR methods are considered to be personalized medicine. This is because CRISPR genome-wide screening identifies drug targets by disrupting genes and observing their effects on disease progression [ 232 ]. This means that CRISPR allows for discovering new therapeutic advancements geared towards specific disease mechanisms [ 232 ]. Additionally, CRISPR has the ability to engineer cells to behave like drug delivery vehicles [ 286 ]. This approach is valuable because CRISPR-edited macrophages can now deliver drugs directly to tumors [ 286 ]. This poses a safe and efficient technique due to minimal side effects on healthy tissues [ 286 ]. Researchers can create libraries that have many varying CRISPR gRNAs that target genes [ 287 ]. These libraries are important because they can be utilized to screen collections of potential drugs [ 287 ]. This CRISPR approach greatly speeds up the drug discovery process. CRISPR-mediated gene editing is also known for its precision when editing genes related to drug discovery in cells or animal models [ 287 ]. Therefore, CRISPR approaches validate drug targets by ensuring the drugs only affect their relevant pathways [ 287 ].

9. CRISPR/Cas and Vaccine Development

The CRISPR/Cas system is a revolutionary technology that has been used to generate recombinant vaccines with greater efficacy, lower cost, and less effort than conventional methods. These traditional techniques, such as gene cloning, bacterial artificial chromosomes (BAC), cosmids, recombinant plasmids, and viral vector-based vaccines, are time consuming, costly, and labor intensive and may have lower efficiency [ 288 ]. In contrast, the CRISPR/Cas system can produce multivalent vaccines that offer long-lasting and protective immunity and have tremendous potential in generating specific B-cells to produce highly potent antibodies against current and future emerging strains of viruses ( Figure 5 A). The CRISPR/Cas system has been used to produce recombinant vaccines for various organisms, such as viruses [ 289 ], bacteria [ 290 ], fungi [ 291 ], plants [ 292 ], and animal cells [ 293 ]. Cas systems can be used to knockout or insert new genes in the cells or viral vector to develop a new generation of vaccines. Indeed, the CRISPR system has been used to edit virus genomes to develop recombinant virus vaccines [ 288 , 294 ]. For example, the Herpesvirus of Turkey (HVT) is a widely used virus vector used for developing vaccines against various diseases, including Marek disease, a common herpes infection in turkeys [ 295 ]. However, the live HVT vector was found to have low immunogenicity, implying that it was not generating a strong immune response. To overcome this issue, researchers have taken advantage of CRISPR/Cas technology to introduce genetic mutations in three genes, gE, gB, and gI, of the HVT vector that displayed a significantly higher level of immunogenicity, indicating a greater potential to stimulate the immune system to fight off infections [ 295 ]. CAS technology was utilized to create a recombinant HVT vaccine vector for infectious bursal disease (IBD) by inserting a VP2 cassette at the UL54-UL56 locus [ 295 ]. The results of this study showed that Cas can effectively generate recombinant vaccine vectors without causing off-target effects [ 295 ]. CRISPR/Cas technology was used in an elegant study to create a multivalent recombinant HVT vector that expresses several antigens. The researchers used CRISPR/Cas to insert gIgD and H8N2 in the HVT-VP2 vector, resulting in a recombinant multivalent vaccine that can target different avian pathogens [ 296 ]. A recent study has utilized the CRISPR/Cas system to create a bivalent recombinant duck enteritis virus (DEV) by inserting hemagglutinin (HA) protein into the UL26 locus of DEV. This study has proven this method to be promising [ 297 ]. Another study has also utilized the CAS system to create trivalent recombinant DEV by inserting premembrane protein (PrM) and glycoprotein E (gE) for duck tembusu virus (DTV) and HA for avian influenza. This was performed to prevent both avian influenza and DTV [ 298 ]. Figure 5 A depicts a strategy that can be used to create recombinant virus vaccine vectors. The use of CRISPR for inserting foreign genes into viral vectors has revolutionized the field by offering an easy, efficient, and time-saving method. By employing CRISPR, researchers successfully inserted the green fluorescence protein into the pseudorabies virus (PRV) genome, enabling the visualization of the virus using microscopy. This breakthrough not only enhances the effectiveness of antiviral drug screening but also streamlines vaccine production [ 299 ]. Furthermore, the CRISPR system facilitated the insertion of a DNA sequence of over 4 kb into the PRV genome for vaccine development [ 300 ]. Using the CRISPR system, a vaccine strain of the PRV was developed by deleting TK (thymidine kinase) and gE genes [ 301 ]. A triple gene deletion of TK/gI and gE in the PRV led to the development of a fully attenuated vaccine that provided protection against infectious PRV strains [ 302 ]. Moreover, the CRISPR/Cas system was used to generate a multivalent recombinant vaccine by deleting virulence factors from the virus genome and inserting a heterologous gene. For instance, CRISPR was employed to knockout the US4 (unique short 4) and TK genes from the infectious laryngotracheitis virus (ILTV) genome and then insert the F (fusion) gene from the Newcastle disease virus (NDV) to form the multivalent recombinant vaccine [ 303 ]. Additionally, in a separate study, CRISPR was utilized to create a recombinant canine distemper virus (CDV) by expressing the F, H, and M (matrix) genes in CDV virus-like particles. The results demonstrated that the recombinant CDV-VLP triggered a robust immune response compared to the parental virus in minks and foxes [ 304 ].

The CAS system has been used to create a recombinant vaccinia virus, which is a popular viral vaccine vector, to produce MAVERICK. MAVERICK is a marker-free vaccinia virus that has been engineered using the CAS system and has been used to create vaccines [ 305 , 306 ]. Moreover, the CAS system has recently been utilized to produce recombinant phages by inserting SARS-CoV2 envelope, spike, and nucleocapsid genes in T4 phage. This process has led to the development of a recombined T4 phage that has shown promising results in inducing a robust T-cell immune response and providing complete protection against virus infection [ 307 , 308 ]. CRISPR/Cas has been utilized in recent research for various applications, including editing the DNA virus genome for vaccine development. The study demonstrated an increase in genome editing through HDR mediation using the NHEJ inhibitor SCR7 in the CRISPR/Cas9 system [ 309 ]. It has been shown that two copies of ICP0 at different locations in the HSV-1 genome could be sequentially edited by the CRISPR/Cas system. These targeted DNA virus editing applications suggest the potential use of the Cas system in developing recombinant virus vaccines [ 309 ]. Furthermore, researchers have used the CRISPR/Cas system to activate latent HIV by targeting the long terminal repeat activator site using dCas9 and the transcriptional activator VP64 [ 310 ]. These studies are valuable for the potential sterile cure of virus infections, as the CRISPR-mediated activation of latent HIV-1 can make patients more receptive to antiviral therapies. Moreover, CRISPR/Cas has been employed to edit parasite DNA to create genetically modified live attenuated whole parasite vaccines [ 311 ]. For instance, it was used to produce a second-generation Leishmania vaccine by creating genetically attenuated Leishmania major, which reduces infectivity and can activate a robust long-term immune response without causing illness [ 311 ]. Leishmania is transmitted by the bite of an infected sand fly.

In response to the emerging SARS-CoV-2 and COVID-19 pandemics, researchers have turned their attention to using the CRISPR/CAS system to target RNA viruses. The Cas system has been used to inhibit the replication of RNA viruses in infected cells [ 312 ]. Cas13 enzymes are part of the class II type VI CRISPR system and have RNA-targeting activities to cleave RNA in human and plant genomes. These CAS nucleases can target both single-strand RNAs (ssRNA) and double-strand RNA viruses [ 312 ]. Notably, studies have reported that the Cas9 system from Francisella can be used to edit ssRNA [ 313 ]. CAS13d has been shown to inhibit SARS-CoV-2 and influenza virus replication in human lung epithelial cells. In this study, the authors identified six crRNAs that can target about 90% of all coronaviruses, marking a significant breakthrough in the field [ 311 ].

B-cells are an essential component in the development of vaccines. They produce immunoglobulins and cytokines that are critical to building immunity and remain present in the body throughout one’s life as plasma cells. The CRISPR/Cas system has wide-ranging applications in engineering B-cells ( Figure 5 B) [ 314 , 315 ]. In fact, studies have shown that the Cas engineering of B-cells can increase their numbers by an incredible 10 folds, with about 80% of PBMC being B-cells [ 314 ]. Primary B-cells can be electroporated between days 3 and 7 post-stimulation, making them highly compatible with this technology [ 314 ]. The CRISPR/Cas technique has been used to introduce frameshift mutations in the papain Fc cleavage site, resulting in Fab secretion instead of complete IgG [ 316 ]. In murine hybridoma, a CRISPR-mediated class switch recombination of 4–11% has been achieved [ 316 ]. Furthermore, CRISPR-mediated gene editing can identify important factors responsible for V(D)J recombination, which is a crucial mechanism for developing immune responses [ 317 ]. Genome editing in activated B-cells has been found to be more efficient due to the upregulation of DNA repair in rapidly proliferating cells [ 318 ]. Scientists have used the CRISPR/Cas system to modify B-cells at the IgH and IgK locus to produce HIV-specific broad-neutralizing antibodies. They found that these engineered B-cells are better in terms of clonal expansion, class switching, and memory retention [ 319 , 320 , 321 ]. In another study, the native heavy chain region of the B-cells was replaced with the PG9 antibody, an anti-HIV antibody. The results showed that human primary B-cells engineered in this way could produce antibodies that bind with gp120 and can undergo affinity maturation [ 322 ]. These studies show that CRISPR/Cas technology can modify B-cells’ genetic material to create highly effective vaccines that target specific diseases. By precisely targeting and modifying B-cells, it is possible to enhance their ability to recognize and fight off pathogens, leading to the development of vaccines with improved efficacy ( Figure 5 B).

10. Conclusions and Future Direction

CRISPR/Cas has been widely used and implemented in many applications for precise genome editing in humans, animals, microbes, plants, and others. It is adapted from the innate immune system of microbials. This technology is highly effective and precise, with minimal off-target effects, making it an excellent tool for correcting genetic mutations. The CRISPR/Cas system has shown great promise in treating genetic diseases, such as sickle cell disease (SCD), thalassemia (TDT), X-linked diseases, eye diseases (ocular gene therapy), and others. CRISPR/Cas has also significantly contributed to cancer research. By knocking out oncogenes and activating tumor suppressor genes, CRISPR/Cas has helped researchers discover novel drug targets for cancer treatments. Moreover, it has been used to delete the genome of oncogenic viruses, such as HPV, which can reduce the incidence of tumor formation in vulnerable populations. Studies have shown that CAR T-cell therapy can effectively improve by inhibiting related genes using CRISPR/Cas editing. CRISPR/Cas can be used to modulate the genome, epigenome, or transcriptome of tumor cells, and it can also be used to produce effective cancer gene therapy. CRISPR/Cas can be delivered using viral and non-viral vectors, with large-capacity viral vectors, such as cytomegalovirus-based vectors, serving as an all-in-one vector delivery for large-sized CRISPR and sgRNA [ 323 , 324 , 325 ]. The CRISPR/Cas system can also be used to cure HIV. Studies have shown that the HIV genome can be removed using CRISPR/Cas for sterilizing the cure of HIV, which will benefit millions of people and prevent disease transmission. Due to high specificity and efficacy, the CRISPR/Cas system has been utilized to generate recombinant vaccines and B-cell engineering to produce more effective vaccines [ 289 , 326 ]. CRISPR/Cas system-based genetic engineering can be used to increase the vaccine yield by removing negative factors that suppress the host immune system. Notably, CRISPR/Cas-engineered vaccines are more effective and require a lower cost and time to generate than conventional vaccines [ 288 , 289 ].

Off-target effects or unintended cutting by CRISPR/Cas is still a major concern when using CRISPR-based gene therapy in clinical and therapeutic applications [ 327 , 328 ]. This unintended cutting can cause harmful mutations or genetic changes that have serious consequences. However, designing highly specific gRNA for target genes and improving the Cas/sgRNA complex can help eliminate the off-target effect of CRISPR. Using the CRISPR-Cas RNP direct delivery to target cells will greatly reduce off-target effects since the RNP degrades after gene editing [ 329 ]. In addition, increasing the delivery efficiency and selecting all-in-one vector delivery methods can also reduce off-target effects and production costs. Currently, most clinical trials use CRISPR/Cas in ex vivo patient-derived cells. These cells are then engineered and injected back into patients, which reduces the potential off-target effects of CRISPR. As most clinical trials for CRISPR-based gene therapy are ongoing, we expect to have a comprehensive understanding of the efficiency and specificity of this technology in patients. One of the major challenges for CRISPR-based gene therapy is delivering it in vivo to specific organs or cells in patients. Due to the lack of robust and effective in vivo delivery tools, this therapy is currently limited to only a few diseases. Another significant challenge is the cost, as approved gene therapies are expensive and are not affordable for most people worldwide. For example, CAR T-cell immunotherapies, like Tisagenlecleucel and Axicabtagene ciloleucel, approved by the US Food and Drug Administration (USFDA), can cost around USD 475,000 and USD 373,000, respectively. In vivo CAR-T or CAR-NK therapy with CRISPR/Cas technology is a promising solution. Another gene therapy named Luxturna ® , used to treat eye diseases caused by mutations in the RPE65 gene, costs approximately EUR 410,550. These gene therapies are not affordable for most people, particularly in Asia, the Middle East, and African countries, where vulnerable populations are predominantly affected. However, CRISPR/Cas may not be effective in treating diseases with multiple mutations, such as cancer, compared to monogenic diseases. Although CRISPR/Cas can correct multiple mutations, off-target effects could lead to the deletion of large DNA fragments or unwanted gene editing. In summary, the potential of CRISPR-based treatments is vast and can play a significant role in biomedicine, with applications in different areas of medicine and biotechnology.

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Author Contributions

Conceptualization, D.K.J. and W.H.; methodology, W.H, D.K.J. and A.S.; software, W.H., D.K.J., A.B., O.I. and A.S.; validation, W.H., D.K.J., A.B., O.I. and A.S.; formal analysis, D.K.J., W.H., A.B., O.I. and A.S.; investigation, D.K.J., W.H., A.B., O.I. and A.S.; resources, D.K.J., A.S. and W.H.; data curation, D.K.J., W.H., A.B., O.I. and A.S.; writing—original draft preparation, A.B., O.I., A.S., D.K.J. and W.H.; writing—D.K.J., W.H., A.B., O.I. and A.S., review and editing, D.K.J., W.H., A.B., O.I., A.S. and A.R.B.; visualization, W.H., D.K.J., A.B., O.I, A.S. and A.R.B.; supervision, D.K.J. and W.H.; project administration, D.K.J. and W.H.; funding acquisition, W.H. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

Ad: adenovirus, AAV: adeno-associated virus, CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats, crRNA: CRISPR RNA, dCas: deactivated Cas, FDA: Food and Drug Administration, HIV: human immunodeficiency virus, LV: lentivirus, LNP: lipid nanoparticle, sgRNA: single guide RNA, DSB: double-stranded break, HDR: homology-directed repair, NHEJ: non-homologous end joining, PAM: protospacer-adjacent motif, TracrRNA: trans-activating CRISPR RNA, ZFN: zinc-finger nuclease.

References

1. Asmamaw M. Zawdie B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing Biologics 2021 15 353 361 10.2147/BTT.S326422 34456559 PMC8388126

2. Ishino Y. Krupovic M. Forterre P. History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology J. Bacteriol. 2018 200 e00580-17 10.1128/JB.00580-17 29358495 PMC5847661

3. Ishino Y. Shinagawa H. Makino K. Amemura M. Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product J. Bacteriol. 1987 169 5429 5433 10.1128/jb.169.12.5429-5433.1987 3316184 PMC213968

4. Janik E. Niemcewicz M. Ceremuga M. Krzowski L. Saluk-Bijak J. Bijak M. Various Aspects of a Gene Editing System-CRISPR-Cas9 Int. J. Mol. Sci. 2020 21 9604 10.3390/ijms21249604 33339441 PMC7767219

5. Zhang B. CRISPR/Cas gene therapy J. Cell. Physiol. 2021 236 2459 2481 10.1002/jcp.30064 32959897

6. Bolotin A. Quinquis B. Sorokin A. Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin Microbiology 2005 151 2551 2561 10.1099/mic.0.28048-0 16079334

7. Mojica F.J. Diez-Villasenor C. Garcia-Martinez J. Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements J. Mol. Evol. 2005 60 174 182 10.1007/s00239-004-0046-3 15791728

8. Barrangou R. Fremaux C. Deveau H. Richards M. Boyaval P. Moineau S. Romero D.A. Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes Science 2007 315 1709 1712 10.1126/science.1138140 17379808

9. Haft D.H. Selengut J. Mongodin E.F. Nelson K.E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes PLoS Comput. Biol. 2005 1 e60 10.1371/journal.pcbi.0010060 16292354 PMC1282333

10. Swarts D.C. Mosterd C. van Passel M.W. Brouns S.J. CRISPR interference directs strand specific spacer acquisition PLoS ONE 2012 7 e35888 10.1371/journal.pone.0035888 22558257 PMC3338789

11. Jinek M. East A. Cheng A. Lin S. Ma E. Doudna J. RNA-programmed genome editing in human cells eLife 2013 2 e00471 10.7554/eLife.00471 23386978 PMC3557905

12. Makarova K.S. Wolf Y.I. Iranzo J. Shmakov S.A. Alkhnbashi O.S. Brouns S.J.J. Charpentier E. Cheng D. Haft D.H. Horvath P. Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants Nat. Rev. Microbiol. 2020 18 67 83 10.1038/s41579-019-0299-x 31857715 PMC8905525

13. Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9 Science 2014 346 1258096 10.1126/science.1258096 25430774

14. Kim J.S. Genome editing comes of age Nat. Protoc. 2016 11 1573 1578 10.1038/nprot.2016.104 27490630

15. Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering Cell 2014 157 1262 1278 10.1016/j.cell.2014.05.010 24906146 PMC4343198

16. Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13 Science 2017 358 1019 1027 10.1126/science.aaq0180 29070703 PMC5793859

17. Harrington L.B. Burstein D. Chen J.S. Paez-Espino D. Ma E. Witte I.P. Cofsky J.C. Kyrpides N.C. Banfield J.F. Doudna J.A. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes Science 2018 362 839 842 10.1126/science.aav4294 30337455 PMC6659742

18. Karvelis T. Bigelyte G. Young J.K. Hou Z. Zedaveinyte R. Budre K. Paulraj S. Djukanovic V. Gasior S. Silanskas A. PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage Nucleic Acids Res. 2020 48 5016 5023 10.1093/nar/gkaa208 32246713 PMC7229846

19. Strecker J. Ladha A. Gardner Z. Schmid-Burgk J.L. Makarova K.S. Koonin E.V. Zhang F. RNA-guided DNA insertion with CRISPR-associated transposases Science 2019 365 48 53 10.1126/science.aax9181 31171706 PMC6659118

20. Dolan A.E. Hou Z. Xiao Y. Gramelspacher M.J. Heo J. Howden S.E. Freddolino P.L. Ke A. Zhang Y. Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas Mol. Cell 2019 74 936 950.e5 10.1016/j.molcel.2019.03.014 30975459 PMC6555677

21. Morisaka H. Yoshimi K. Okuzaki Y. Gee P. Kunihiro Y. Sonpho E. Xu H. Sasakawa N. Naito Y. Nakada S. CRISPR-Cas3 induces broad and unidirectional genome editing in human cells Nat. Commun. 2019 10 5302 10.1038/s41467-019-13226-x 31811138 PMC6897959

22. Hidalgo-Cantabrana C. Goh Y.J. Pan M. Sanozky-Dawes R. Barrangou R. Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus Proc. Natl. Acad. Sci. USA 2019 116 15774 15783 10.1073/pnas.1905421116 31341082 PMC6690032

23. Mei Y. Wang Y. Chen H. Sun Z.S. Ju X.D. Recent Progress in CRISPR/Cas9 Technology J. Genet. Genom. 2016 43 63 75 10.1016/j.jgg.2016.01.001 26924689

24. Shao M. Xu T.R. Chen C.S. The big bang of genome editing technology: Development and application of the CRISPR/Cas9 system in disease animal models Dongwuxue Yanjiu 2016 37 191 204 10.13918/j.issn.2095-8137.2016.4.191 27469250 PMC4980067

25. Nishimasu H. Ran F.A. Hsu P.D. Konermann S. Shehata S.I. Dohmae N. Ishitani R. Zhang F. Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA Cell 2014 156 935 949 10.1016/j.cell.2014.02.001 24529477 PMC4139937

26. Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science 2012 337 816 821 10.1126/science.1225829 22745249 PMC6286148

27. Gasiunas G. Barrangou R. Horvath P. Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Proc. Natl. Acad. Sci. USA 2012 109 E2579 E2586 10.1073/pnas.1208507109 22949671 PMC3465414

28. Cong L. Zhang F. Genome engineering using CRISPR-Cas9 system Methods Mol. Biol. 2015 1239 197 217 10.1007/978-1-4939-1862-1_10 25408407

29. Hirano S. Nishimasu H. Ishitani R. Nureki O. Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9 Mol. Cell 2016 61 886 894 10.1016/j.molcel.2016.02.018 26990991

30. Ceasar S.A. Rajan V. Prykhozhij S.V. Berman J.N. Ignacimuthu S. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9 Biochim. Biophys. Acta 2016 1863 2333 2344 10.1016/j.bbamcr.2016.06.009 27350235

31. Haapaniemi E. Botla S. Persson J. Schmierer B. Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response Nat. Med. 2018 24 927 930 10.1038/s41591-018-0049-z 29892067

32. Roman-Rodriguez F.J. Ugalde L. Alvarez L. Diez B. Ramirez M.J. Risueno C. Corton M. Bogliolo M. Bernal S. March F. NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia Cell Stem Cell 2019 25 607 621.e7 10.1016/j.stem.2019.08.016 31543367

33. Lino C.A. Harper J.C. Carney J.P. Timlin J.A. Delivering CRISPR: A review of the challenges and approaches Drug Deliv. 2018 25 1234 1257 10.1080/10717544.2018.1474964 29801422 PMC6058482

34. Komor A.C. Badran A.H. Liu D.R. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes Cell 2017 168 20 36 10.1016/j.cell.2016.10.044 27866654 PMC5235943

35. Kim Y.B. Komor A.C. Levy J.M. Packer M.S. Zhao K.T. Liu D.R. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions Nat. Biotechnol. 2017 35 371 376 10.1038/nbt.3803 28191901 PMC5388574

36. Koblan L.W. Doman J.L. Wilson C. Levy J.M. Tay T. Newby G.A. Maianti J.P. Raguram A. Liu D.R. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction Nat. Biotechnol. 2018 36 843 846 10.1038/nbt.4172 29813047 PMC6126947

37. Li X. Wang Y. Liu Y. Yang B. Wang X. Wei J. Lu Z. Zhang Y. Wu J. Huang X. Base editing with a Cpf1-cytidine deaminase fusion Nat. Biotechnol. 2018 36 324 327 10.1038/nbt.4102 29553573

38. Gaudelli N.M. Komor A.C. Rees H.A. Packer M.S. Badran A.H. Bryson D.I. Liu D.R. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage Nature 2017 551 464 471 10.1038/nature24644 29160308 PMC5726555

39. Huang T.P. Zhao K.T. Miller S.M. Gaudelli N.M. Oakes B.L. Fellmann C. Savage D.F. Liu D.R. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors Nat. Biotechnol. 2019 37 626 631 10.1038/s41587-019-0134-y 31110355 PMC6551276

40. Richter M.F. Zhao K.T. Eton E. Lapinaite A. Newby G.A. Thuronyi B.W. Wilson C. Koblan L.W. Zeng J. Bauer D.E. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity Nat. Biotechnol. 2020 38 883 891 10.1038/s41587-020-0453-z 32433547 PMC7357821

41. Li T. Yang Y. Qi H. Cui W. Zhang L. Fu X. He X. Liu M. Li P.F. Yu T. CRISPR/Cas9 therapeutics: Progress and prospects Signal Transduct. Target. Ther. 2023 8 36 10.1038/s41392-023-01309-7 36646687 PMC9841506

42. Kurt I.C. Zhou R. Iyer S. Garcia S.P. Miller B.R. Langner L.M. Grunewald J. Joung J.K. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells Nat. Biotechnol. 2021 39 41 46 10.1038/s41587-020-0609-x 32690971 PMC7854778

43. Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression Cell 2013 152 1173 1183 10.1016/j.cell.2013.02.022 23452860 PMC3664290

44. Gilbert L.A. Horlbeck M.A. Adamson B. Villalta J.E. Chen Y. Whitehead E.H. Guimaraes C. Panning B. Ploegh H.L. Bassik M.C. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation Cell 2014 159 647 661 10.1016/j.cell.2014.09.029 25307932 PMC4253859

45. Maeder M.L. Linder S.J. Cascio V.M. Fu Y. Ho Q.H. Joung J.K. CRISPR RNA-guided activation of endogenous human genes Nat. Methods 2013 10 977 979 10.1038/nmeth.2598 23892898 PMC3794058

46. Bernstein B.E. Meissner A. Lander E.S. The mammalian epigenome Cell 2007 128 669 681 10.1016/j.cell.2007.01.033 17320505

47. Pulecio J. Verma N. Mejia-Ramirez E. Huangfu D. Raya A. CRISPR/Cas9-Based Engineering of the Epigenome Cell Stem Cell 2017 21 431 447 10.1016/j.stem.2017.09.006 28985525 PMC6205890

48. Chen B. Gilbert L.A. Cimini B.A. Schnitzbauer J. Zhang W. Li G.W. Park J. Blackburn E.H. Weissman J.S. Qi L.S. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system Cell 2013 155 1479 1491 10.1016/j.cell.2013.12.001 24360272 PMC3918502

49. Qin P. Parlak M. Kuscu C. Bandaria J. Mir M. Szlachta K. Singh R. Darzacq X. Yildiz A. Adli M. Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9 Nat. Commun. 2017 8 14725 10.1038/ncomms14725 28290446 PMC5424063

50. Shmakov S. Smargon A. Scott D. Cox D. Pyzocha N. Yan W. Abudayyeh O.O. Gootenberg J.S. Makarova K.S. Wolf Y.I. Diversity and evolution of class 2 CRISPR-Cas systems Nat. Rev. Microbiol. 2017 15 169 182 10.1038/nrmicro.2016.184 28111461 PMC5851899

51. Pyzocha N.K. Chen S. Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications ACS Chem. Biol. 2018 13 347 356 10.1021/acschembio.7b00800 29121460 PMC6768076

52. Perez Rojo F. Nyman R.K.M. Johnson A.A.T. Navarro M.P. Ryan M.H. Erskine W. Kaur P. CRISPR-Cas systems: Ushering in the new genome editing era Bioengineered 2018 9 214 221 10.1080/21655979.2018.1470720 29968520 PMC6067892

53. Zhang J. Liu J. Yang W. Cui M. Dai B. Dong Y. Yang J. Zhang X. Liu D. Liang H. Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats Theriogenology 2019 132 1 11 10.1016/j.theriogenology.2019.03.029 30981084

54. Ates I. Rathbone T. Stuart C. Bridges P.H. Cottle R.N. Delivery Approaches for Therapeutic Genome Editing and Challenges Genes 2020 11 1113 10.3390/genes11101113 32977396 PMC7597956

55. Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Multiplex genome engineering using CRISPR/Cas systems Science 2013 339 819 823 10.1126/science.1231143 23287718 PMC3795411

56. Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9 Science 2013 339 823 826 10.1126/science.1232033 23287722 PMC3712628

57. Das T. Anand U. Pal T. Mandal S. Kumar M. Radha Gopalakrishnan A.V. Lastra J.M.P. Dey A. Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches Biotechnol. Bioeng. 2023 120 1215 1228 10.1002/bit.28344 36740587

58. Ayanoglu F.B. Elcin A.E. Elcin Y.M. Bioethical issues in genome editing by CRISPR-Cas9 technology Turk. J. Biol. 2020 44 110 120 10.3906/biy-1912-52 32256147 PMC7129066

59. Eid A. Mahfouz M.M. Genome editing: The road of CRISPR/Cas9 from bench to clinic Exp. Mol. Med. 2016 48 e265 10.1038/emm.2016.111 27741224 PMC5099421

60. Yi L. Li J. CRISPR-Cas9 therapeutics in cancer: Promising strategies and present challenges Biochim. Biophys. Acta 2016 1866 197 207 10.1016/j.bbcan.2016.09.002 27641687

61. Singh P. Ali S.A. Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals Vet. Sci. 2021 8 122 10.3390/vetsci8070122 34209174 PMC8309983

62. Jaganathan D. Ramasamy K. Sellamuthu G. Jayabalan S. Venkataraman G. CRISPR for Crop Improvement: An Update Review Front. Plant Sci. 2018 9 985 10.3389/fpls.2018.00985 30065734 PMC6056666

63. King A. A CRISPR edit for heart disease Nature 2018 555 S23 S25 10.1038/d41586-018-02482-4 32094978

64. Guan L. Han Y. Yang C. Lu S. Du J. Li H. Lin J. CRISPR-Cas9-Mediated Gene Therapy in Neurological Disorders Mol. Neurobiol. 2022 59 968 982 10.1007/s12035-021-02638-w 34813019

65. Binnie A. Fernandes E. Almeida-Lousada H. de Mello R.A. Castelo-Branco P. CRISPR-based strategies in infectious disease diagnosis and therapy Infection 2021 49 377 385 10.1007/s15010-020-01554-w 33393066 PMC7779109

66. Baddeley H.J.E. Isalan M. The Application of CRISPR/Cas Systems for Antiviral Therapy Front. Genome Ed. 2021 3 745559 10.3389/fgeed.2021.745559 34723245 PMC8549726

67. Mayorga-Ramos A. Zuniga-Miranda J. Carrera-Pacheco S.E. Barba-Ostria C. Guaman L.P. CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance ACS Infect. Dis. 2023 9 1283 1302 10.1021/acsinfecdis.2c00649 37347230 PMC10353011

68. Tao S. Chen H. Li N. Liang W. The Application of the CRISPR-Cas System in Antibiotic Resistance Infect. Drug Resist. 2022 15 4155 4168 10.2147/IDR.S370869 35942309 PMC9356603

69. Abdelnour S.A. Xie L. Hassanin A.A. Zuo E.R. Lu Y.Q. The Potential of CRISPR/Cas9 Gene Editing as a Treatment Strategy for Inherited Diseases Front. Cell Dev. Biol. 2021 9 699597 10.3389/fcell.2021.699597 34977000 PMC8715006

70. Liu P.P. Huang L. Song C.Q. Editorial: Genome editing applications of CRISPR/Cas9 in metabolic diseases, hormonal system and cancer research Front. Endocrinol. 2023 14 1256966 10.3389/fendo.2023.1256966 PMC10441539 37608792

71. Lakhawat S.S. Malik N. Kumar V. Kumar S. Sharma P.K. Implications of CRISPR-Cas9 in Developing Next Generation Biofuel: A Mini-review Curr. Protein Pept. Sci. 2022 23 574 584 10.2174/1389203723666220907110310 36082852

72. Xu R. Yang Y. Qin R. Li H. Qiu C. Li L. Wei P. Yang J. Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice J. Genet. Genom. 2016 43 529 532 10.1016/j.jgg.2016.07.003 27543262

73. Zsogon A. Cermak T. Naves E.R. Notini M.M. Edel K.H. Weinl S. Freschi L. Voytas D.F. Kudla J. Peres L.E.P. De novo domestication of wild tomato using genome editing Nat. Biotechnol. 2018 36 1211 1216 10.1038/nbt.4272 30272678

74. Rodriguez-Leal D. Lemmon Z.H. Man J. Bartlett M.E. Lippman Z.B. Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing Cell 2017 171 470 480.e8 10.1016/j.cell.2017.08.030 28919077

75. Nishihara M. Higuchi A. Watanabe A. Tasaki K. Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri BMC Plant Biol. 2018 18 331 10.1186/s12870-018-1539-3 30518324 PMC6280492

76. Xu Z.S. Yang Q.Q. Feng K. Xiong A.S. Changing Carrot Color: Insertions in DcMYB7 Alter the Regulation of Anthocyanin Biosynthesis and Modification Plant Physiol. 2019 181 195 207 10.1104/pp.19.00523 31213511 PMC6716245

77. Yu Q.H. Wang B. Li N. Tang Y. Yang S. Yang T. Xu J. Guo C. Yan P. Wang Q. CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines Sci. Rep. 2017 7 11874 10.1038/s41598-017-12262-1 28928381 PMC5605656

78. Hu C. Sheng O. Deng G. He W. Dong T. Yang Q. Dou T. Li C. Gao H. Liu S. CRISPR/Cas9-mediated genome editing of MaACO1 (aminocyclopropane-1-carboxylate oxidase 1) promotes the shelf life of banana fruit Plant Biotechnol. J. 2021 19 654 656 10.1111/pbi.13534 33369835 PMC8051599

79. Uluisik S. Chapman N.H. Smith R. Poole M. Adams G. Gillis R.B. Besong T.M. Sheldon J. Stiegelmeyer S. Perez L. Genetic improvement of tomato by targeted control of fruit softening Nat. Biotechnol. 2016 34 950 952 10.1038/nbt.3602 27454737

80. Elitzur T. Yakir E. Quansah L. Zhangjun F. Vrebalov J. Khayat E. Giovannoni J.J. Friedman H. Banana MaMADS Transcription Factors Are Necessary for Fruit Ripening and Molecular Tools to Promote Shelf-Life and Food Security Plant Physiol. 2016 171 380 391 10.1104/pp.15.01866 26956665 PMC4854685

81. Dong O.X. Yu S. Jain R. Zhang N. Duong P.Q. Butler C. Li Y. Lipzen A. Martin J.A. Barry K.W. Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9 Nat. Commun. 2020 11 1178 10.1038/s41467-020-14981-y 32132530 PMC7055238

82. Nuss P. Anxiety disorders and GABA neurotransmission: A disturbance of modulation Neuropsychiatr. Dis. Treat. 2015 11 165 175 10.2147/NDT.S58841 25653526 PMC4303399

83. Li R. Li R. Li X. Fu D. Zhu B. Tian H. Luo Y. Zhu H. Multiplexed CRISPR/Cas9-mediated metabolic engineering of gamma-aminobutyric acid levels in Solanum lycopersicum Plant Biotechnol. J. 2018 16 415 427 10.1111/pbi.12781 28640983 PMC5787826

84. Menchaca A. Dos Santos-Neto P.C. Mulet A.P. Crispo M. CRISPR in livestock: From editing to printing Theriogenology 2020 150 247 254 10.1016/j.theriogenology.2020.01.063 32088034 PMC7102594

85. Whitworth K.M. Rowland R.R. Ewen C.L. Trible B.R. Kerrigan M.A. Cino-Ozuna A.G. Samuel M.S. Lightner J.E. McLaren D.G. Mileham A.J. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus Nat. Biotechnol. 2016 34 20 22 10.1038/nbt.3434 26641533

86. Burkard C. Opriessnig T. Mileham A.J. Stadejek T. Ait-Ali T. Lillico S.G. Whitelaw C.B.A. Archibald A.L. Pigs Lacking the Scavenger Receptor Cysteine-Rich Domain 5 of CD163 Are Resistant to Porcine Reproductive and Respiratory Syndrome Virus 1 Infection J. Virol. 2018 92 e00415-18 10.1128/JVI.00415-18 29925651 PMC6069206

87. Hammond A. Galizi R. Kyrou K. Simoni A. Siniscalchi C. Katsanos D. Gribble M. Baker D. Marois E. Russell S. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae Nat. Biotechnol. 2016 34 78 83 10.1038/nbt.3439 26641531 PMC4913862

88. Galizi R. Hammond A. Kyrou K. Taxiarchi C. Bernardini F. O’Loughlin S.M. Papathanos P.A. Nolan T. Windbichler N. Crisanti A. A CRISPR-Cas9 sex-ratio distortion system for genetic control Sci. Rep. 2016 6 31139 10.1038/srep31139 27484623 PMC4971495

89. Wang S.W. Gao C. Zheng Y.M. Yi L. Lu J.C. Huang X.Y. Cai J.B. Zhang P.F. Cui Y.H. Ke A.W. Current applications and future perspective of CRISPR/Cas9 gene editing in cancer Mol. Cancer 2022 21 57 10.1186/s12943-022-01518-8 35189910 PMC8862238

90. Wilbie D. Walther J. Mastrobattista E. Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing Acc. Chem. Res. 2019 52 1555 1564 10.1021/acs.accounts.9b00106 31099553 PMC6584901

91. Bessis N. GarciaCozar F.J. Boissier M.C. Immune responses to gene therapy vectors: Influence on vector function and effector mechanisms Gene Ther. 2004 11 (Suppl. 1) S10 S17 10.1038/sj.gt.3302364 15454952

92. Butt M.H. Zaman M. Ahmad A. Khan R. Mallhi T.H. Hasan M.M. Khan Y.H. Hafeez S. Massoud E.E.S. Rahman M.H. Appraisal for the Potential of Viral and Nonviral Vectors in Gene Therapy: A Review Genes 2022 13 1370 10.3390/genes13081370 36011281 PMC9407213

93. Mendell J.R. Al-Zaidy S.A. Rodino-Klapac L.R. Goodspeed K. Gray S.J. Kay C.N. Boye S.L. Boye S.E. George L.A. Salabarria S. Current Clinical Applications of In Vivo Gene Therapy with AAVs Mol. Ther. 2021 29 464 488 10.1016/j.ymthe.2020.12.007 33309881 PMC7854298

94. Luo J. Luo Y. Sun J. Zhou Y. Zhang Y. Yang X. Adeno-associated virus-mediated cancer gene therapy: Current status Cancer Lett. 2015 356 347 356 10.1016/j.canlet.2014.10.045 25444906 PMC4259838

95. Li C. Samulski R.J. Engineering adeno-associated virus vectors for gene therapy Nat. Rev. Genet. 2020 21 255 272 10.1038/s41576-019-0205-4 32042148

96. Nelson C.E. Gersbach C.A. Engineering Delivery Vehicles for Genome Editing Annu. Rev. Chem. Biomol. Eng. 2016 7 637 662 10.1146/annurev-chembioeng-080615-034711 27146557

97. Lundstrom K. Viral Vectors in Gene Therapy Diseases 2018 6 42 10.3390/diseases6020042 29883422 PMC6023384

98. Kuzmin D.A. Shutova M.V. Johnston N.R. Smith O.P. Fedorin V.V. Kukushkin Y.S. van der Loo J.C.M. Johnstone E.C. The clinical landscape for AAV gene therapies Nat. Rev. Drug Discov. 2021 20 173 174 10.1038/d41573-021-00017-7 33495615

99. Burdett T. Nuseibeh S. Changing trends in the development of AAV-based gene therapies: A meta-analysis of past and present therapies Gene Ther. 2023 30 323 335 10.1038/s41434-022-00363-0 36089633

100. Wang G. Genome Editing for Cystic Fibrosis Cells 2023 12 1555 10.3390/cells12121555 37371025 PMC10297084

101. Senis E. Fatouros C. Grosse S. Wiedtke E. Niopek D. Mueller A.K. Borner K. Grimm D. CRISPR/Cas9-mediated genome engineering: An adeno-associated viral (AAV) vector toolbox Biotechnol. J. 2014 9 1402 1412 10.1002/biot.201400046 25186301

102. Wang D. Zhang F. Gao G. CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors Cell 2020 181 136 150 10.1016/j.cell.2020.03.023 32243786 PMC7236621

103. Dong J.Y. Fan P.D. Frizzell R.A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus Hum. Gene Ther. 1996 7 2101 2112 10.1089/hum.1996.7.17-2101 8934224

104. Yang Y. Wang L. Bell P. McMenamin D. He Z. White J. Yu H. Xu C. Morizono H. Musunuru K. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice Nat. Biotechnol. 2016 34 334 338 10.1038/nbt.3469 26829317 PMC4786489

105. Esvelt K.M. Mali P. Braff J.L. Moosburner M. Yaung S.J. Church G.M. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing Nat. Methods 2013 10 1116 1121 10.1038/nmeth.2681 24076762 PMC3844869

106. Ran F.A. Cong L. Yan W.X. Scott D.A. Gootenberg J.S. Kriz A.J. Zetsche B. Shalem O. Wu X. Makarova K.S. In vivo genome editing using Staphylococcus aureus Cas9 Nature 2015 520 186 191 10.1038/nature14299 25830891 PMC4393360

107. Kumar N. Stanford W. de Solis C. Aradhana Abraham N.D. Dao T.J. Thaseen S. Sairavi A. Gonzalez C.U. Ploski J.E. The Development of an AAV-Based CRISPR SaCas9 Genome Editing System That Can Be Delivered to Neurons in vivo and Regulated via Doxycycline and Cre-Recombinase Front. Mol. Neurosci. 2018 11 413 10.3389/fnmol.2018.00413 30483052 PMC6243075

108. Hino T. Omura S.N. Nakagawa R. Togashi T. Takeda S.N. Hiramoto T. Tasaka S. Hirano H. Tokuyama T. Uosaki H. An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis Cell 2023 186 4920 4935.e23 10.1016/j.cell.2023.08.031 37776859

109. Vigna E. Naldini L. Lentiviral vectors: Excellent tools for experimental gene transfer and promising candidates for gene therapy J. Gene Med. 2000 2 308 316 10.1002/1521-2254(200009/10)2:5<308::AID-JGM131>3.0.CO;2-3 11045424

110. Kantor B. Bailey R.M. Wimberly K. Kalburgi S.N. Gray S.J. Methods for gene transfer to the central nervous system Adv. Genet. 2014 87 125 197 10.1016/B978-0-12-800149-3.00003-2 25311922 PMC4519829

111. Lee S. Kim Y.Y. Ahn H.J. Systemic delivery of CRISPR/Cas9 to hepatic tumors for cancer treatment using altered tropism of lentiviral vector Biomaterials 2021 272 120793 10.1016/j.biomaterials.2021.120793 33836291

112. Ciuffi A. Mechanisms governing lentivirus integration site selection Curr. Gene Ther. 2008 8 419 429 10.2174/156652308786848021 19075625

113. Wanisch K. Yanez-Munoz R.J. Integration-deficient lentiviral vectors: A slow coming of age Mol. Ther. 2009 17 1316 1332 10.1038/mt.2009.122 19491821 PMC2835240

114. Zhang S. Shen J. Li D. Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing Theranostics 2021 11 614 648 10.7150/thno.47007 33391496 PMC7738854

115. Gong J. Chung T.H. Zheng J. Zheng H. Chang L.J. Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A J. Biol. Chem. 2021 297 101397 10.1016/j.jbc.2021.101397 34774524 PMC8649223

116. Dahl M. Doyle A. Olsson K. Mansson J.E. Marques A.R.A. Mirzaian M. Aerts J.M. Ehinger M. Rothe M. Modlich U. Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice Mol. Ther. 2015 23 835 844 10.1038/mt.2015.16 25655314 PMC4427872

117. Pack D.W. Hoffman A.S. Pun S. Stayton P.S. Design and development of polymers for gene delivery Nat. Rev. Drug Discov. 2005 4 581 593 10.1038/nrd1775 16052241

118. Sercombe L. Veerati T. Moheimani F. Wu S.Y. Sood A.K. Hua S. Advances and Challenges of Liposome Assisted Drug Delivery Front. Pharmacol. 2015 6 286 10.3389/fphar.2015.00286 26648870 PMC4664963

119. Finn J.D. Smith A.R. Patel M.C. Shaw L. Youniss M.R. van Heteren J. Dirstine T. Ciullo C. Lescarbeau R. Seitzer J. A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing Cell Rep. 2018 22 2227 2235 10.1016/j.celrep.2018.02.014 29490262

120. Liu J.J. Orlova N. Oakes B.L. Ma E. Spinner H.B. Baney K.L.M. Chuck J. Tan D. Knott G.J. Harrington L.B. CasX enzymes comprise a distinct family of RNA-guided genome editors Nature 2019 566 218 223 10.1038/s41586-019-0908-x 30718774 PMC6662743

121. Pausch P. Al-Shayeb B. Bisom-Rapp E. Tsuchida C.A. Li Z. Cress B.F. Knott G.J. Jacobsen S.E. Banfield J.F. Doudna J.A. CRISPR-CasPhi from huge phages is a hypercompact genome editor Science 2020 369 333 337 10.1126/science.abb1400 32675376 PMC8207990

122. Wu Z. Zhang Y. Yu H. Pan D. Wang Y. Wang Y. Li F. Liu C. Nan H. Chen W. Programmed genome editing by a miniature CRISPR-Cas12f nuclease Nat. Chem. Biol. 2021 17 1132 1138 10.1038/s41589-021-00868-6 34475565

123. Strecker J. Jones S. Koopal B. Schmid-Burgk J. Zetsche B. Gao L. Makarova K.S. Koonin E.V. Zhang F. Engineering of CRISPR-Cas12b for human genome editing Nat. Commun. 2019 10 212 10.1038/s41467-018-08224-4 30670702 PMC6342934

124. Kim D.Y. Lee J.M. Moon S.B. Chin H.J. Park S. Lim Y. Kim D. Koo T. Ko J.H. Kim Y.S. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus Nat. Biotechnol. 2022 40 94 102 10.1038/s41587-021-01009-z 34475560 PMC8763643

125. Xu X. Chemparathy A. Zeng L. Kempton H.R. Shang S. Nakamura M. Qi L.S. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing Mol. Cell 2021 81 4333 4345.e4 10.1016/j.molcel.2021.08.008 34480847

126. Bigelyte G. Young J.K. Karvelis T. Budre K. Zedaveinyte R. Djukanovic V. Van Ginkel E. Paulraj S. Gasior S. Jones S. Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells Nat. Commun. 2021 12 6191 10.1038/s41467-021-26469-4 34702830 PMC8548392

127. Xiao R. Li Z. Wang S. Han R. Chang L. Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease Nucleic Acids Res. 2021 49 4120 4128 10.1093/nar/gkab179 33764415 PMC8053092

128. Takeda S.N. Nakagawa R. Okazaki S. Hirano H. Kobayashi K. Kusakizako T. Nishizawa T. Yamashita K. Nishimasu H. Nureki O. Structure of the miniature type V-F CRISPR-Cas effector enzyme Mol. Cell 2021 81 558 570.e3 10.1016/j.molcel.2020.11.035 33333018

129. Wu T. Liu C. Zou S. Lyu R. Yang B. Yan H. Zhao M. Tang W. An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity Nat. Chem. Biol. 2023 19 1384 1393 10.1038/s41589-023-01380-9 37400536 PMC10625714

130. Heckl D. Kowalczyk M.S. Yudovich D. Belizaire R. Puram R.V. McConkey M.E. Thielke A. Aster J.C. Regev A. Ebert B.L. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing Nat. Biotechnol. 2014 32 941 946 10.1038/nbt.2951 24952903 PMC4160386

131. Israel M.A. Yuan S.H. Bardy C. Reyna S.M. Mu Y. Herrera C. Hefferan M.P. Van Gorp S. Nazor K.L. Boscolo F.S. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells Nature 2012 482 216 220 10.1038/nature10821 22278060 PMC3338985

132. Xue H.Y. Ji L.J. Gao A.M. Liu P. He J.D. Lu X.J. CRISPR-Cas9 for medical genetic screens: Applications and future perspectives J. Med. Genet. 2016 53 91 97 10.1136/jmedgenet-2015-103409 26673779

133. Novak L.C. Chou J. Colic M. Bristow C.A. Hart T. PICKLES v3: The updated database of pooled in vitro CRISPR knockout library essentiality screens Nucleic Acids Res. 2023 51 D1117 D1121 10.1093/nar/gkac982 36350677 PMC9825567

134. Pardee K. Green A.A. Takahashi M.K. Braff D. Lambert G. Lee J.W. Ferrante T. Ma D. Donghia N. Fan M. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components Cell 2016 165 1255 1266 10.1016/j.cell.2016.04.059 27160350

135. Kellner M.J. Koob J.G. Gootenberg J.S. Abudayyeh O.O. Zhang F. SHERLOCK: Nucleic acid detection with CRISPR nucleases Nat. Protoc. 2019 14 2986 3012 10.1038/s41596-019-0210-2 31548639 PMC6956564

136. Gootenberg J.S. Abudayyeh O.O. Kellner M.J. Joung J. Collins J.J. Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 Science 2018 360 439 444 10.1126/science.aaq0179 29449508 PMC5961727

137. Chen J.S. Ma E. Harrington L.B. Da Costa M. Tian X. Palefsky J.M. Doudna J.A. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity Science 2018 360 436 439 10.1126/science.aar6245 29449511 PMC6628903

138. Park C.Y. Kim D.H. Son J.S. Sung J.J. Lee J. Bae S. Kim J.H. Kim D.W. Kim J.S. Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9 Cell Stem Cell 2015 17 213 220 10.1016/j.stem.2015.07.001 26212079

139. Morishige S. Mizuno S. Ozawa H. Nakamura T. Mazahery A. Nomura K. Seki R. Mouri F. Osaki K. Yamamura K. CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs Int. J. Hematol. 2020 111 225 233 10.1007/s12185-019-02765-0 31664646

140. Xie F. Ye L. Chang J.C. Beyer A.I. Wang J. Muench M.O. Kan Y.W. Seamless gene correction of beta-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac Genome Res. 2014 24 1526 1533 10.1101/gr.173427.114 25096406 PMC4158758

141. Antony J.S. Latifi N. Haque A. Lamsfus-Calle A. Daniel-Moreno A. Graeter S. Baskaran P. Weinmann P. Mezger M. Handgretinger R. Gene correction of HBB mutations in CD34(+) hematopoietic stem cells using Cas9 mRNA and ssODN donors Mol. Cell. Pediatr. 2018 5 9 10.1186/s40348-018-0086-1 30430274 PMC6236008

142. Schwank G. Koo B.K. Sasselli V. Dekkers J.F. Heo I. Demircan T. Sasaki N. Boymans S. Cuppen E. van der Ent C.K. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients Cell Stem Cell 2013 13 653 658 10.1016/j.stem.2013.11.002 24315439

143. Fan Z. Perisse I.V. Cotton C.U. Regouski M. Meng Q. Domb C. Van Wettere A.J. Wang Z. Harris A. White K.L. A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene JCI Insight 2018 3 e123529 10.1172/jci.insight.123529 30282831 PMC6237476

144. Rabinowitz R. Kadair A. Ben-Zur T. Michaelson D. Offen D. Apoe4 Allele Specific Knockout Using a Synthetic Cas9 Variant as a Potential Gene Therapy Approach for Alzheimer’s Disease Cytotherapy 2019 21 E7 10.1016/j.jcyt.2019.04.022

145. Shin J.W. Kim K.H. Chao M.J. Atwal R.S. Gillis T. MacDonald M.E. Gusella J.F. Lee J.M. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9 Hum. Mol. Genet. 2016 25 4566 4576 10.1093/hmg/ddw286 28172889 PMC6078600

146. Vermilyea S.C. Babinski A. Tran N. To S. Guthrie S. Kluss J.H. Schmidt J.K. Wiepz G.J. Meyer M.G. Murphy M.E. In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson’s Disease in Common Marmosets Sci. Rep. 2020 10 3447 10.1038/s41598-020-60273-2 32103062 PMC7044232

147. Shao Y.J. Wang L.R. Guo N.N. Wang S.F. Yang L. Li Y.J. Wang M.S. Yin S.M. Han H.H. Zeng L. Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats J. Biol. Chem. 2018 293 6883 6892 10.1074/jbc.RA117.000347 29507093 PMC5936814

148. Shimo T. Hosoki K. Nakatsuji Y. Yokota T. Obika S. A novel human muscle cell model of Duchenne muscular dystrophy created by CRISPR/Cas9 and evaluation of antisense-mediated exon skipping J. Hum. Genet. 2018 63 365 375 10.1038/s10038-017-0400-0 29339778

149. Anzalone A.V. Randolph P.B. Davis J.R. Sousa A.A. Koblan L.W. Levy J.M. Chen P.J. Wilson C. Newby G.A. Raguram A. Search-and-replace genome editing without double-strand breaks or donor DNA Nature 2019 576 149 157 10.1038/s41586-019-1711-4 31634902 PMC6907074

150. Hu J. Chen L. Yin J. Yin H.C. Huang Y.N. Tian J.J. Hyperactivity, Memory Defects, and Craniofacial Abnormalities in Zebrafish Mutant Larvae Behav. Genet. 2020 50 152 160 10.1007/s10519-020-09995-7 32048109

151. Yu W.H. Mookherjee S. Chaitankar V. Hiriyanna S. Kim J.W. Brooks M. Ataeijannati Y. Sun X. Dong L.J. Li T.S. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice Nat. Commun. 2017 8 14716 10.1038/ncomms14716 28291770 PMC5355895

152. Wu Y.X. Liang D. Wang Y.H. Bai M.Z. Tang W. Bao S.M. Yan Z.Q. Li D.S. Li J.S. Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9 Cell Stem Cell 2013 13 659 662 10.1016/j.stem.2013.10.016 24315440

153. Zhao Z.L. Shi L. Zhang W. Han J.S. Zhang S.H. Fu Z.X. Cai J.H. CRISPR knock out of programmed cell death protein 1 enhances anti-tumor activity of cytotoxic T lymphocytes Oncotarget 2018 9 5208 5215 10.18632/oncotarget.23730 29435173 PMC5797044

154. Huang X.S. Wang Y. Yan W. Smith C. Ye Z.H. Wang J. Gao Y.X. Mendelsohn L. Cheng L.Z. Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated From Patient iPSCs After Genome Editing of the Sickle Point Mutation Stem Cells 2015 33 1470 1479 10.1002/stem.1969 25702619 PMC4628786

155. Lin H.F. Li G. Peng X.W. Deng A.M. Ye L. Shi L. Wang T.M. He J. The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses Front. Cell. Infect. Microbiol. 2021 11 590989 10.3389/fcimb.2021.590989 34513721 PMC8430244

156. Teng F. Cui T. Feng G. Guo L. Xu K. Gao Q. Li T. Li J. Zhou Q. Li W. Repurposing CRISPR-Cas12b for mammalian genome engineering Cell Discov. 2018 4 63 10.1038/s41421-018-0069-3 30510770 PMC6255809

157. Joung J. Ladha A. Saito M. Kim N.G. Woolley A.E. Segel M. Barretto R.P.J. Ranu A. Macrae R.K. Faure G. Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing N. Engl. J. Med. 2020 383 1492 1494 10.1056/NEJMc2026172 32937062 PMC7510942

158. Cyranoski D. Chinese scientists to pioneer first human CRISPR trial Nature 2016 535 476 477 10.1038/nature.2016.20302 27466105

159. Platt R.J. Chen S. Zhou Y. Yim M.J. Swiech L. Kempton H.R. Dahlman J.E. Parnas O. Eisenhaure T.M. Jovanovic M. CRISPR-Cas9 knockin mice for genome editing and cancer modeling Cell 2014 159 440 455 10.1016/j.cell.2014.09.014 25263330 PMC4265475

160. Zhan T. Rindtorff N. Betge J. Ebert M.P. Boutros M. CRISPR/Cas9 for cancer research and therapy Semin. Cancer Biol. 2019 55 106 119 10.1016/j.semcancer.2018.04.001 29673923

161. Vermersch E. Jouve C. Hulot J.S. CRISPR/Cas9 gene-editing strategies in cardiovascular cells Cardiovasc. Res. 2020 116 894 907 10.1093/cvr/cvz250 31584620

162. Newby G.A. Yen J.S. Woodard K.J. Mayuranathan T. Lazzarotto C.R. Li Y. Sheppard-Tillman H. Porter S.N. Yao Y. Mayberry K. Base editing of haematopoietic stem cells rescues sickle cell disease in mice Nature 2021 595 295 302 10.1038/s41586-021-03609-w 34079130 PMC8266759

163. Heidenreich M. Zhang F. Applications of CRISPR-Cas systems in neuroscience Nat. Rev. Neurosci. 2016 17 36 44 10.1038/nrn.2015.2 26656253 PMC4899966

164. Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Nature 2016 533 420 424 10.1038/nature17946 27096365 PMC4873371

165. Hu J.H. Miller S.M. Geurts M.H. Tang W.X. Chen L.W. Sun N. Zeina C.M. Gao X. Rees H.A. Lin Z. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity Nature 2018 556 57 63 10.1038/nature26155 29512652 PMC5951633

166. Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B.T. Shmakov S. Makarova K.S. Semenova E. Minakhin L. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector Science 2016 353 aaf5573 10.1126/science.aaf5573 27256883 PMC5127784

167. Hirano H. Gootenberg J.S. Horii T. Abudayyeh O.O. Kimura M. Hsu P.D. Nakane T. Ishitani R. Hatada I. Zhang F. Structure and Engineering of Francisella novicida Cas9 Cell 2016 164 950 961 10.1016/j.cell.2016.01.039 26875867 PMC4899972

168. Kim E. Koo T. Park S.W. Kim D. Kim K. Cho H.Y. Song D.W. Lee K.J. Jung M.H. Kim S. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni Nat. Commun. 2017 8 14500 10.1038/ncomms14500 28220790 PMC5473640

169. Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system Cell 2015 163 759 771 10.1016/j.cell.2015.09.038 26422227 PMC4638220

170. Fonfara I. Richter H. Bratovic M. Le Rhun A. Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA Nature 2016 532 517 521 10.1038/nature17945 27096362

171. Casini A. Olivieri M. Petris G. Montagna C. Reginato G. Maule G. Lorenzin F. Prandi D. Romanel A. Demichelis F. A highly specific SpCas9 variant is identified by screening in yeast Nat. Biotechnol. 2018 36 265 271 10.1038/nbt.4066 29431739 PMC6066108

172. Hou Z.G. Zhang Y. Propson N.E. Howden S.E. Chu L.F. Sontheimer E.J. Thomson J.A. Efficient genome engineering in human pluripotent stem cells using Cas9 from Proc. Natl. Acad. Sci. USA 2013 110 15644 15649 10.1073/pnas.1313587110 23940360 PMC3785731

173. Garneau J.E. Dupuis M.E. Villion M. Romero D.A. Barrangou R. Boyaval P. Fremaux C. Horvath P. Magadán A.H. Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Nature 2010 468 67 71 10.1038/nature09523 21048762

174. Magadán A.H. Dupuis M.E. Villion M. Moineau S. Cleavage of Phage DNA by the CRISPR3-Cas System PLoS ONE 2012 7 e40913 10.1371/journal.pone.0040913 22911717 PMC3401199

175. Slaymaker I.M. Gao L.Y. Zetsche B. Scott D.A. Yan W.X. Zhang F. Rationally engineered Cas9 nucleases with improved specificity Science 2016 351 84 88 10.1126/science.aad5227 26628643 PMC4714946

176. Kleinstiver B.P. Prew M.S. Tsai S.Q. Topkar V.V. Nguyen N.T. Zheng Z.L. Gonzales A.P.W. Li Z.Y. Peterson R.T. Yeh J.R.J. Engineered CRISPR-Cas9 nucleases with altered PAM specificities Nature 2015 523 481 485 10.1038/nature14592 26098369 PMC4540238

177. Kleinstiver B.P. Pattanayak V. Prew M.S. Tsai S.Q. Nguyen N.T. Zheng Z.L. Joung J.K. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects Nature 2016 529 490 495 10.1038/nature16526 26735016 PMC4851738

178. Nishimasu H. Shi X. Ishiguro S. Gao L.Y. Hirano S. Okazaki S. Noda T. Abudayyeh O.O. Gootenberg J.S. Mori H. Engineered CRISPR-Cas9 nuclease with expanded targeting space Science 2018 361 1259 1262 10.1126/science.aas9129 30166441 PMC6368452

179. Kleinstiver B.P. Prew M.S. Tsai S.Q. Nguyen N.T. Topkar V.V. Zheng Z.L. Joung J.K. Broadening the targeting range of CRISPR-Cas9 by modifying PAM recognition Nat. Biotechnol. 2015 33 1293 1298 10.1038/nbt.3404 26524662 PMC4689141

180. Chen J.S. Dagdas Y.S. Kleinstiver B.P. Welch M.M. Sousa A.A. Harrington L.B. Sternberg S.H. Joung J.K. Yildiz A. Doudna J.A. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy Nature 2017 550 407 410 10.1038/nature24268 28931002 PMC5918688

181. Lee J.K. Jeong E. Lee J. Jung M. Shin E. Kim Y.H. Lee K. Jung I. Kim D. Kim S. Directed evolution of CRISPR-Cas9 to increase its specificity Nat. Commun. 2018 9 3048 10.1038/s41467-018-05477-x 30082838 PMC6078992

182. Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants Science 2020 368 290 296 10.1126/science.aba8853 32217751 PMC7297043

183. Miller S.M. Wang T. Randolph P.B. Arbab M. Shen M.W. Huang T.P. Matuszek Z. Newby G.A. Rees H.A. Liu D.R. Continuous evolution of SpCas9 variants compatible with non-G PAMs Nat. Biotechnol. 2020 38 471 481 10.1038/s41587-020-0412-8 32042170 PMC7145744

184. Kong X. Zhang H. Li G. Wang Z. Kong X. Wang L. Xue M. Zhang W. Wang Y. Lin J. Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing Nat. Commun. 2023 14 2046 10.1038/s41467-023-37829-7 37041195 PMC10090079

185. Su M. Li F. Wang Y. Gao Y. Lan W. Shao Z. Zhu C. Tang N. Gan J. Wu Z. Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity Nat. Chem. Biol. 2024 20 180 189 10.1038/s41589-023-01420-4 37697004

186. Chen W. Ma J. Wu Z. Wang Z. Zhang H. Fu W. Pan D. Shi J. Ji Q. Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors Mol. Cell 2023 83 2768 2780.e6 10.1016/j.molcel.2023.06.014 37402371

187. Das S. Bano S. Kapse P. Kundu G.C. CRISPR based therapeutics: A new paradigm in cancer precision medicine Mol. Cancer 2022 21 85 10.1186/s12943-022-01552-6 35337340 PMC8953071

188. Song X. Liu C. Wang N. Huang H. He S. Gong C. Wei Y. Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy Adv. Drug Deliv. Rev. 2021 168 158 180 10.1016/j.addr.2020.04.010 32360576

189. Kazemizadeh H. Kashefizadeh A. CRISPR-Cas9-mediated gene therapy in lung cancer Clin. Transl. Oncol. 2023 25 1156 1166 10.1007/s12094-022-03039-8 36495467

190. Brentjens R.J. Davila M.L. Riviere I. Park J. Wang X. Cowell L.G. Bartido S. Stefanski J. Taylor C. Olszewska M. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia Sci. Transl. Med. 2013 5 177ra138 10.1126/scitranslmed.3005930 23515080 PMC3742551

191. Park J.H. Riviere I. Gonen M. Wang X. Senechal B. Curran K.J. Sauter C. Wang Y. Santomasso B. Mead E. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia N. Engl. J. Med. 2018 378 449 459 10.1056/NEJMoa1709919 29385376 PMC6637939

192. Braendstrup P. Levine B.L. Ruella M. The long road to the first FDA-approved gene therapy: Chimeric antigen receptor T cells targeting CD19 Cytotherapy 2020 22 57 69 10.1016/j.jcyt.2019.12.004 32014447 PMC7036015

193. Fraietta J.A. Lacey S.F. Orlando E.J. Pruteanu-Malinici I. Gohil M. Lundh S. Boesteanu A.C. Wang Y. O’Connor R.S. Hwang W.T. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia Nat. Med. 2018 24 563 571 10.1038/s41591-018-0010-1 29713085 PMC6117613

194. Xu Y. Zhang M. Ramos C.A. Durett A. Liu E. Dakhova O. Liu H. Creighton C.J. Gee A.P. Heslop H.E. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15 Blood 2014 123 3750 3759 10.1182/blood-2014-01-552174 24782509 PMC4055922

195. Hurton L.V. Singh H. Najjar A.M. Switzer K.C. Mi T. Maiti S. Olivares S. Rabinovich B. Huls H. Forget M.A. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells Proc. Natl. Acad. Sci. USA 2016 113 E7788 E7797 10.1073/pnas.1610544113 27849617 PMC5137758

196. Kasakovski D. Xu L. Li Y. T cell senescence and CAR-T cell exhaustion in hematological malignancies J. Hematol. Oncol. 2018 11 91 10.1186/s13045-018-0629-x 29973238 PMC6032767

197. Fedorov V.D. Themeli M. Sadelain M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses Sci. Transl. Med. 2013 5 215ra172 10.1126/scitranslmed.3006597 24337479 PMC4238416

198. Zhang Y. Zhang X. Cheng C. Mu W. Liu X. Li N. Wei X. Liu X. Xia C. Wang H. CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells Front. Med. 2017 11 554 562 10.1007/s11684-017-0543-6 28625015

199. Zhang W. Shi L. Zhao Z. Du P. Ye X. Li D. Cai Z. Han J. Cai J. Disruption of CTLA-4 expression on peripheral blood CD8 + T cell enhances anti-tumor efficacy in bladder cancer Cancer Chemother. Pharmacol. 2019 83 911 920 10.1007/s00280-019-03800-x 30848330

200. Liu X. Zhang Y. Cheng C. Cheng A.W. Zhang X. Li N. Xia C. Wei X. Liu X. Wang H. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells Cell Res. 2017 27 154 157 10.1038/cr.2016.142 27910851 PMC5223227

201. Dimitri A. Herbst F. Fraietta J.A. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing Mol. Cancer 2022 21 78 10.1186/s12943-022-01559-z 35303871 PMC8932053

202. Jung I.Y. Kim Y.Y. Yu H.S. Lee M. Kim S. Lee J. CRISPR/Cas9-Mediated Knockout of DGK Improves Antitumor Activities of Human T Cells Cancer Res. 2018 78 4692 4703 10.1158/0008-5472.CAN-18-0030 29967261

203. Sterner R.M. Sakemura R. Cox M.J. Yang N. Khadka R.H. Forsman C.L. Hansen M.J. Jin F. Ayasoufi K. Hefazi M. GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts Blood 2019 133 697 709 10.1182/blood-2018-10-881722 30463995 PMC6376281

204. Bonifant C.L. Jackson H.J. Brentjens R.J. Curran K.J. Toxicity and management in CAR T-cell therapy Mol. Ther. Oncol. 2016 3 16011 10.1038/mto.2016.11 PMC5008265 27626062

205. Eyquem J. Mansilla-Soto J. Giavridis T. van der Stegen S.J. Hamieh M. Cunanan K.M. Odak A. Gonen M. Sadelain M. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection Nature 2017 543 113 117 10.1038/nature21405 28225754 PMC5558614

206. Huang L. Liao Z. Liu Z. Chen Y. Huang T. Xiao H. Application and Prospect of CRISPR/Cas9 Technology in Reversing Drug Resistance of Non-Small Cell Lung Cancer Front. Pharmacol. 2022 13 900825 10.3389/fphar.2022.900825 35620280 PMC9127258

207. Sorolla A. Parisi E. Sorolla M.A. Marques M. Porcel J.M. Applications of CRISPR technology to lung cancer research Eur. Respir. J. 2022 59 2102610 10.1183/13993003.02610-2021 34737228

208. Stefanoudakis D. Kathuria-Prakash N. Sun A.W. Abel M. Drolen C.E. Ashbaugh C. Zhang S. Hui G. Tabatabaei Y.A. Zektser Y. The Potential Revolution of Cancer Treatment with CRISPR Technology Cancers 2023 15 1813 10.3390/cancers15061813 36980699 PMC10046289

209. Camidge D.R. Pao W. Sequist L.V. Acquired resistance to TKIs in solid tumours: Learning from lung cancer Nat. Rev. Clin. Oncol. 2014 11 473 481 10.1038/nrclinonc.2014.104 24981256

210. Robichaux J.P. Elamin Y.Y. Tan Z. Carter B.W. Zhang S. Liu S. Li S. Chen T. Poteete A. Estrada-Bernal A. Mechanisms and clinical activity of an EGFR and HER2 exon 20-selective kinase inhibitor in non-small cell lung cancer Nat. Med. 2018 24 638 646 10.1038/s41591-018-0007-9 29686424 PMC5964608

211. Tang H. Shrager J.B. CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer: A personalized molecular surgical therapy EMBO Mol. Med. 2016 8 83 85 10.15252/emmm.201506006 26747090 PMC4734839

212. Gao Q. Ouyang W. Kang B. Han X. Xiong Y. Ding R. Li Y. Wang F. Huang L. Chen L. Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression Theranostics 2020 10 5137 5153 10.7150/thno.42325 32308773 PMC7163449

213. Wu X. Huang H. Yu B. Zhang J. A Blue Light-Inducible CRISPR-Cas9 System for Inhibiting Progression of Melanoma Cells Front. Mol. Biosci. 2020 7 606593 10.3389/fmolb.2020.606593 33330635 PMC7710612

214. Yao S. He Z. Chen C. CRISPR/Cas9-Mediated Genome Editing of Epigenetic Factors for Cancer Therapy Hum. Gene Ther. 2015 26 463 471 10.1089/hum.2015.067 26075804

215. Tsherniak A. Vazquez F. Montgomery P.G. Weir B.A. Kryukov G. Cowley G.S. Gill S. Harrington W.F. Pantel S. Krill-Burger J.M. Defining a Cancer Dependency Map Cell 2017 170 564 576.e16 10.1016/j.cell.2017.06.010 28753430 PMC5667678

216. Zhu G.D. Yu J. Sun Z.Y. Chen Y. Zheng H.M. Lin M.L. Ou-Yang S. Liu G.L. Zhang J.W. Shao F.M. Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma Cell Death Dis. 2021 12 724 10.1038/s41419-021-04000-3 34290231 PMC8295287

217. Huang K. Liu X. Li Y. Wang Q. Zhou J. Wang Y. Dong F. Yang C. Sun Z. Fang C. Genome-Wide CRISPR-Cas9 Screening Identifies NF-kappaB/E2F6 Responsible for EGFRvIII-Associated Temozolomide Resistance in Glioblastoma Adv. Sci. 2019 6 1900782 10.1002/advs.201900782 31508283 PMC6724471

218. Zou Y. Sun X. Yang Q. Zheng M. Shimoni O. Ruan W. Wang Y. Zhang D. Yin J. Huang X. Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy Sci. Adv. 2022 8 eabm8011 10.1126/sciadv.abm8011 35442747 PMC9020780

219. Zhao N. Weng S. Liu Z. Xu H. Ren Y. Guo C. Liu L. Zhang Z. Ji Y. Han X. CRISPR-Cas9 identifies growth-related subtypes of glioblastoma with therapeutical significance through cell line knockdown BMC Cancer 2023 23 749 10.1186/s12885-023-11131-7 37580710 PMC10424363

220. Mao X.Y. Dai J.X. Zhou H.H. Liu Z.Q. Jin W.L. Brain tumor modeling using the CRISPR/Cas9 system: State of the art and view to the future Oncotarget 2016 7 33461 33471 10.18632/oncotarget.8075 26993776 PMC5078110

221. Begagic E. Beculic H. Duzic N. Dzidic-Krivic A. Pugonja R. Muharemovic A. Jaganjac B. Salkovic N. Sefo H. Pojskic M. CRISPR/Cas9-Mediated Gene Therapy for Glioblastoma: A Scoping Review Biomedicines 2024 12 238 10.3390/biomedicines12010238 38275409 PMC10813360

222. Frangoul H. Altshuler D. Cappellini M.D. Chen Y.S. Domm J. Eustace B.K. Foell J. de la Fuente J. Grupp S. Handgretinger R. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia N. Engl. J. Med. 2021 384 252 260 10.1056/NEJMoa2031054 33283989

223. Sharma A. Boelens J.J. Cancio M. Hankins J.S. Bhad P. Azizy M. Lewandowski A. Zhao X. Chitnis S. Peddinti R. CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease N. Engl. J. Med. 2023 389 820 832 10.1056/NEJMoa2215643 37646679 PMC10947132

224. Park S.H. Bao G. CRISPR/Cas9 gene editing for curing sickle cell disease Transfus. Apher. Sci. 2021 60 103060 10.1016/j.transci.2021.103060 33455878 PMC8049447

225. Ma L. Yang S. Peng Q. Zhang J. Zhang J. CRISPR/Cas9-based gene-editing technology for sickle cell disease Gene 2023 874 147480 10.1016/j.gene.2023.147480 37182559

226. Pinhas A. Zhou D.B. Otero-Marquez O. Castanos Toral M.V. Migacz J.V. Glassberg J. Rosen R.B. Chui T.Y.P. Efficacy of CRISPR-Based Gene Editing in a Sickle Cell Disease Patient as Measured through the Eye Case Rep. Hematol. 2022 2022 6079631 10.1155/2022/6079631 36046774 PMC9424027

227. Maule G. Arosio D. Cereseto A. Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing Int. J. Mol. Sci. 2020 21 3903 10.3390/ijms21113903 32486152 PMC7313467

228. Hodges C.A. Conlon R.A. Delivering on the promise of gene editing for cystic fibrosis Genes. Dis. 2019 6 97 108 10.1016/j.gendis.2018.11.005 31193992 PMC6545485

229. Lee J.A. Cho A. Huang E.N. Xu Y. Quach H. Hu J. Wong A.P. Gene therapy for cystic fibrosis: New tools for precision medicine J. Transl. Med. 2021 19 452 10.1186/s12967-021-03099-4 34717671 PMC8556969

230. Lee M.H. Shin J.I. Yang J.W. Lee K.H. Cha D.H. Hong J.B. Park Y. Choi E. Tizaoui K. Koyanagi A. Genome Editing Using CRISPR-Cas9 and Autoimmune Diseases: A Comprehensive Review Int. J. Mol. Sci. 2022 23 1337 10.3390/ijms23031337 35163260 PMC8835887

231. Xiao D. Zhang W. Wang Q. Li X. Zhang Y. Rasouli J. Casella G. Ciric B. Curtis M. Rostami A. CRISPR-mediated rapid generation of neural cell-specific knockout mice facilitates research in neurophysiology and pathology Mol. Ther. Methods Clin. Dev. 2021 20 755 764 10.1016/j.omtm.2021.02.012 33738329 PMC7940702

232. Kendirli A. de la Rosa C. Lammle K.F. Eglseer K. Bauer I.J. Kavaka V. Winklmeier S. Zhuo L. Wichmann C. Gerdes L.A. A genome-wide in vivo CRISPR screen identifies essential regulators of T cell migration to the CNS in a multiple sclerosis model Nat. Neurosci. 2023 26 1713 1725 10.1038/s41593-023-01432-2 37709997 PMC10545543

233. Hussein M. Molina M.A. Berkhout B. Herrera-Carrillo E. A CRISPR-Cas Cure for HIV/AIDS Int. J. Mol. Sci. 2023 24 1563 10.3390/ijms24021563 36675077 PMC9863116

234. Kaminski R. Chen Y. Fischer T. Tedaldi E. Napoli A. Zhang Y. Karn J. Hu W. Khalili K. Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing Sci. Rep. 2016 6 22555 10.1038/srep22555 26939770 PMC4778041

235. Hu W. Kaminski R. Yang F. Zhang Y. Cosentino L. Li F. Luo B. Alvarez-Carbonell D. Garcia-Mesa Y. Karn J. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection Proc. Natl. Acad. Sci. USA 2014 111 11461 11466 10.1073/pnas.1405186111 25049410 PMC4128125

236. Dash P.K. Kaminski R. Bella R. Su H. Mathews S. Ahooyi T.M. Chen C. Mancuso P. Sariyer R. Ferrante P. Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice Nat. Commun. 2019 10 2753 10.1038/s41467-019-10366-y 31266936 PMC6606613

237. Fan M. Berkhout B. Herrera-Carrillo E. A combinatorial CRISPR-Cas12a attack on HIV DNA Mol. Ther. Methods Clin. Dev. 2022 25 43 51 10.1016/j.omtm.2022.02.010 35356755 PMC8933334

238. Yin C. Zhang T. Qu X. Zhang Y. Putatunda R. Xiao X. Li F. Xiao W. Zhao H. Dai S. In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models Mol. Ther. 2017 25 1168 1186 10.1016/j.ymthe.2017.03.012 28366764 PMC5417847

239. Ophinni Y. Inoue M. Kotaki T. Kameoka M. CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures Sci. Rep. 2018 8 7784 10.1038/s41598-018-26190-1 29773895 PMC5958087

240. Zhu W. Lei R. Le Duff Y. Li J. Guo F. Wainberg M.A. Liang C. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA Retrovirology 2015 12 22 10.1186/s12977-015-0150-z 25808449 PMC4359768

241. Yin L. Zhao F. Sun H. Wang Z. Huang Y. Zhu W. Xu F. Mei S. Liu X. Zhang D. CRISPR-Cas13a Inhibits HIV-1 Infection Mol. Ther. Nucleic Acids 2020 21 147 155 10.1016/j.omtn.2020.05.030 32585623 PMC7321785

242. Yin C. Zhang T. Li F. Yang F. Putatunda R. Young W.B. Khalili K. Hu W. Zhang Y. Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS AIDS 2016 30 1163 1174 10.1097/QAD.0000000000001079 26990633 PMC4851589

243. Swiech L. Heidenreich M. Banerjee A. Habib N. Li Y. Trombetta J. Sur M. Zhang F. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9 Nat. Biotechnol. 2015 33 102 106 10.1038/nbt.3055 25326897 PMC4492112

244. Zuris J.A. Thompson D.B. Shu Y. Guilinger J.P. Bessen J.L. Hu J.H. Maeder M.L. Joung J.K. Chen Z.Y. Liu D.R. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo Nat. Biotechnol. 2015 33 73 80 10.1038/nbt.3081 25357182 PMC4289409

245. Yin H. Xue W. Chen S. Bogorad R.L. Benedetti E. Grompe M. Koteliansky V. Sharp P.A. Jacks T. Anderson D.G. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype Nat. Biotechnol. 2014 32 551 553 10.1038/nbt.2884 24681508 PMC4157757

246. Yin H. Song C.Q. Dorkin J.R. Zhu L.J. Li Y. Wu Q. Park A. Yang J. Suresh S. Bizhanova A. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo Nat. Biotechnol. 2016 34 328 333 10.1038/nbt.3471 26829318 PMC5423356

247. Kaminski R. Bella R. Yin C. Otte J. Ferrante P. Gendelman H.E. Li H. Booze R. Gordon J. Hu W. Excision of HIV-1 DNA by gene editing: A proof-of-concept in vivo study Gene Ther. 2016 23 690 695 10.1038/gt.2016.41 27194423 PMC4974122

248. Mancuso P. Chen C. Kaminski R. Gordon J. Liao S. Robinson J.A. Smith M.D. Liu H. Sariyer I.K. Sariyer R. CRISPR based editing of SIV proviral DNA in ART treated non-human primates Nat. Commun. 2020 11 6065 10.1038/s41467-020-19821-7 33247091 PMC7695718

249. Bhowmik R. Chaubey B. CRISPR/Cas9: A tool to eradicate HIV-1 AIDS Res. Ther. 2022 19 58 10.1186/s12981-022-00483-y 36457057 PMC9713993

250. Xiao Q. Guo D. Chen S. Application of CRISPR/Cas9-Based Gene Editing in HIV-1/AIDS Therapy Front. Cell. Infect. Microbiol. 2019 9 69 10.3389/fcimb.2019.00069 30968001 PMC6439341

251. Dash P.K. Chen C. Kaminski R. Su H. Mancuso P. Sillman B. Zhang C. Liao S. Sravanam S. Liu H. CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice Proc. Natl. Acad. Sci. USA 2023 120 e2217887120 10.1073/pnas.2217887120 37126704 PMC10175831

252. Vasconcelos Komninakis S. Domingues W. Saeed Sanabani S. Angelo Folgosi V. Neves Barbosa I. Casseb J. CRISPR/CAS as a Powerful Tool for Human Immunodeficiency Virus Cure: A Review AIDS Res. Hum. Retroviruses 2024 Online ahead of print 10.1089/AID.2022.0148 38164106

253. Wei Z. Bodnar B. Zhao R.T. Xiao Q. Saribas S. Wang X. Ho W.Z. Hu W. Human iPSC-derived brain organoids: A 3D mini-brain model for studying HIV infection Exp. Neurol. 2023 364 114386 10.1016/j.expneurol.2023.114386 36934866 PMC10149614

254. Bogerd H.P. Kornepati A.V. Marshall J.B. Kennedy E.M. Cullen B.R. Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators Proc. Natl. Acad. Sci. USA 2015 112 E7249 E7256 10.1073/pnas.1516305112 26668372 PMC4703010

255. Ehrke-Schulz E. Schiwon M. Leitner T. David S. Bergmann T. Liu J. Ehrhardt A. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes Sci. Rep. 2017 7 17113 10.1038/s41598-017-17180-w 29215041 PMC5719366

256. Xu L. Yang H. Gao Y. Chen Z. Xie L. Liu Y. Liu Y. Wang X. Li H. Lai W. CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo Mol. Ther. 2017 25 1782 1789 10.1016/j.ymthe.2017.04.027 28527722 PMC5542791

257. Li C. Guan X. Du T. Jin W. Wu B. Liu Y. Wang P. Hu B. Griffin G.E. Shattock R.J. Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9 J. Gen. Virol. 2015 96 2381 2393 10.1099/vir.0.000139 25854553

258. Nahmad A.D. Lazzarotto C.R. Zelikson N. Kustin T. Tenuta M. Huang D. Reuveni I. Nataf D. Raviv Y. Horovitz-Fried M. In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice Nat. Biotechnol. 2022 40 1241 1249 10.1038/s41587-022-01328-9 35681059 PMC7613293

259. Liu Z. Chen S. Jin X. Wang Q. Yang K. Li C. Xiao Q. Hou P. Liu S. Wu S. Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4 + T cells from HIV-1 infection Cell Biosci. 2017 7 47 10.1186/s13578-017-0174-2 28904745 PMC5591563

260. Zhang Y. Ozono S. Yao W. Tobiume M. Yamaoka S. Kishigami S. Fujita H. Tokunaga K. CRISPR-mediated activation of endogenous BST-2/tetherin expression inhibits wild-type HIV-1 production Sci. Rep. 2019 9 3134 10.1038/s41598-019-40003-z 30816279 PMC6395588

261. Wang G. Zhao N. Berkhout B. Das A.T. A Combinatorial CRISPR-Cas9 Attack on HIV-1 DNA Extinguishes All Infectious Provirus in Infected T Cell Cultures Cell Rep. 2016 17 2819 2826 10.1016/j.celrep.2016.11.057 27974196

262. Nguyen H. Wilson H. Jayakumar S. Kulkarni V. Kulkarni S. Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System Viruses 2021 13 1850 10.3390/v13091850 34578431 PMC8473377

263. Kaminski R. Chen Y. Salkind J. Bella R. Young W.B. Ferrante P. Karn J. Malcolm T. Hu W. Khalili K. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy Sci. Rep. 2016 6 31527 10.1038/srep31527 27528385 PMC4985742

264. Okee M. Bayiyana A. Musubika C. Joloba M.L. Ashaba-Katabazi F. Bagaya B. Wayengera M. In Vitro Transduction and Target-Mutagenesis Efficiency of HIV-1 pol Gene Targeting ZFN and CRISPR/Cas9 Delivered by Various Plasmids and/or Vectors: Toward an HIV Cure AIDS Res. Hum. Retroviruses 2018 34 88 102 10.1089/AID.2017.0234 29183134

265. Ophinni Y. Miki S. Hayashi Y. Kameoka M. Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape Viruses 2020 12 1223 10.3390/v12111223 33126728 PMC7693572

266. Herskovitz J. Hasan M. Patel M. Blomberg W.R. Cohen J.D. Machhi J. Shahjin F. Mosley R.L. McMillan J. Kevadiya B.D. CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination eBioMedicine 2021 73 103678 10.1016/j.ebiom.2021.103678 34774454 PMC8633974

267. Gao Z. Fan M. Das A.T. Herrera-Carrillo E. Berkhout B. Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA Nucleic Acids Res. 2020 48 5527 5539 10.1093/nar/gkaa226 32282899 PMC7261156

268. Xu L. Wang J. Liu Y. Xie L. Su B. Mou D. Wang L. Liu T. Wang X. Zhang B. CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia N. Engl. J. Med. 2019 381 1240 1247 10.1056/NEJMoa1817426 31509667

269. Stadtmauer E.A. Fraietta J.A. Davis M.M. Cohen A.D. Weber K.L. Lancaster E. Mangan P.A. Kulikovskaya I. Gupta M. Chen F. CRISPR-engineered T cells in patients with refractory cancer Science 2020 367 eaba7365 10.1126/science.aba7365 32029687 PMC11249135

270. Blau N. Shen N. Carducci C. Molecular genetics and diagnosis of phenylketonuria: State of the art Expert. Rev. Mol. Diagn. 2014 14 655 671 10.1586/14737159.2014.923760 24882081

271. Villiger L. Grisch-Chan H.M. Lindsay H. Ringnalda F. Pogliano C.B. Allegri G. Fingerhut R. Haberle J. Matos J. Robinson M.D. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice Nat. Med. 2018 24 1519 1525 10.1038/s41591-018-0209-1 30297904

272. Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. Guembe L. Moreno-Cermeno A. Corrales F.J. Prieto J. Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy J. Hepatol. 2016 64 419 426 10.1016/j.jhep.2015.09.014 26409215

273. Wang D. Chen L. Li C. Long Q. Yang Q. Huang A. Tang H. CRISPR/Cas9 delivery by NIR-responsive biomimetic nanoparticles for targeted HBV therapy J. Nanobiotechnol. 2022 20 27 10.1186/s12951-021-01233-4 PMC8740473 34991617

274. Sin Y.Y. Price P.R. Ballantyne L.L. Funk C.D. Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency Sci. Rep. 2017 7 2585 10.1038/s41598-017-02927-2 28566761 PMC5451454

275. Sung J.J. Park C.Y. Leem J.W. Cho M.S. Kim D.W. Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs Exp. Mol. Med. 2019 51 1 9 10.1038/s12276-019-0243-1 PMC6470126 30996250

276. Park C.Y. Sung J.J. Cho S.R. Kim J. Kim D.W. Universal Correction of Blood Coagulation Factor VIII in Patient-Derived Induced Pluripotent Stem Cells Using CRISPR/Cas9 Stem Cell Rep. 2019 12 1242 1249 10.1016/j.stemcr.2019.04.016 31105049 PMC6565751

277. Hodges P.E. Rosenberg L.E. The spfash mouse: A missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing Proc. Natl. Acad. Sci. USA 1989 86 4142 4146 10.1073/pnas.86.11.4142 2471197 PMC287405

278. Zhao D. Liu J. Wang M. Zhang X. Zhou M. Epidemiology of cardiovascular disease in China: Current features and implications Nat. Rev. Cardiol. 2019 16 203 212 10.1038/s41569-018-0119-4 30467329

279. Tadros R.O. Tang G.H.L. Barnes H.J. Mousavi I. Kovacic J.C. Faries P. Olin J.W. Marin M.L. Adams D.H. Optimal Treatment of Uncomplicated Type B Aortic Dissection: JACC Review Topic of the Week J. Am. Coll. Cardiol. 2019 74 1494 1504 10.1016/j.jacc.2019.07.063 31514953

280. Mehta L.S. Beckie T.M. DeVon H.A. Grines C.L. Krumholz H.M. Johnson M.N. Lindley K.J. Vaccarino V. Wang T.Y. Watson K.E. Acute Myocardial Infarction in Women: A Scientific Statement From the American Heart Association Circulation 2016 133 916 947 10.1161/CIR.0000000000000351 26811316

281. Oka T. Akazawa H. Naito A.T. Komuro I. Angiogenesis and cardiac hypertrophy: Maintenance of cardiac function and causative roles in heart failure Circ. Res. 2014 114 565 571 10.1161/CIRCRESAHA.114.300507 24481846

282. Madsen A. Hoppner G. Krause J. Hirt M.N. Laufer S.D. Schweizer M. Tan W.L.W. Mosqueira D. Anene-Nzelu C.G. Lim I. An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility Circulation 2020 142 1562 1578 10.1161/CIRCULATIONAHA.119.044444 32885664 PMC7566310

283. Zhang X. Xu C. Gao S. Li P. Kong Y. Li T. Li Y. Xu F.J. Du J. CRISPR/Cas9 Delivery Mediated with Hydroxyl-Rich Nanosystems for Gene Editing in Aorta Adv. Sci. 2019 6 1900386 10.1002/advs.201900386 31380173 PMC6662060

284. Zhang X. Jin H. Huang X. Chaurasiya B. Dong D. Shanley T.P. Zhao Y.Y. Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA Cell Rep. 2022 38 110196 10.1016/j.celrep.2021.110196 34986352 PMC8769807

285. McLean B. Istadi A. Clack T. Vankan M. Schramek D. Neely G.G. Pajic M. A CRISPR Path to Finding Vulnerabilities and Solving Drug Resistance: Targeting the Diverse Cancer Landscape and Its Ecosystem Adv. Genet. 2022 3 2200014 10.1002/ggn2.202200014 36911295 PMC9993475

286. Tiedt R. King F.J. Stamm C. Niederst M.J. Delach S. Zumstein-Mecker S. Meltzer J. Mulford I.J. Labrot E. Engstler B.S. Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors Cell Rep. 2023 42 112297 10.1016/j.celrep.2023.112297 36961816

287. Fellmann C. Gowen B.G. Lin P.C. Doudna J.A. Corn J.E. Cornerstones of CRISPR-Cas in drug discovery and therapy Nat. Rev. Drug Discov. 2017 16 89 100 10.1038/nrd.2016.238 28008168 PMC5459481

288. Li H. Yang Y. Hong W. Huang M. Wu M. Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: Mechanisms, advances and prospects Signal Transduct. Target. Ther. 2020 5 1 10.1038/s41392-019-0089-y 32296011 PMC6946647

289. Bhujbal S. Bhujbal R. Giram P. An overview: CRISPR/Cas-based gene editing for viral vaccine development Expert. Rev. Vaccines 2022 21 1581 1593 10.1080/14760584.2022.2112952 35959589

290. Ou B. Jiang B. Jin D. Yang Y. Zhang M. Zhang D. Zhao H. Xu M. Song H. Wu W. Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo ACS Synth. Biol. 2020 9 412 426 10.1021/acssynbio.9b00430 31944664

291. Mitsui R. Yamada R. Ogino H. CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals World J. Microbiol. Biotechnol. 2019 35 111 10.1007/s11274-019-2688-8 31280424

292. Cao Y. Zhou H. Zhou X. Li F. Control of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity Front. Microbiol. 2020 11 593700 10.3389/fmicb.2020.593700 33193268 PMC7649272

293. van der Sanden S.M. Wu W. Dybdahl-Sissoko N. Weldon W.C. Brooks P. O’Donnell J. Jones L.P. Brown C. Tompkins S.M. Oberste M.S. Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: The Polio End Game J. Virol. 2016 90 1694 1704 10.1128/JVI.01464-15 26581994 PMC4733985

294. Yin H. Li Z. Zhang J. Huang J. Kang H. Tian J. Qu L. Construction of a US7/US8/UL23/US3-deleted recombinant pseudorabies virus and evaluation of its pathogenicity in dogs Vet. Microbiol. 2020 240 108543 10.1016/j.vetmic.2019.108543 31902487

295. Tang N. Zhang Y. Pedrera M. Chang P. Baigent S. Moffat K. Shen Z. Nair V. Yao Y. A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system Vaccine 2018 36 716 722 10.1016/j.vaccine.2017.12.025 29269155 PMC5783714

296. Tang N. Zhang Y. Sadigh Y. Moffat K. Shen Z. Nair V. Yao Y. Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing Vaccines 2020 8 97 10.3390/vaccines8010097 32098149 PMC7157232

297. Chang P. Yao Y. Tang N. Sadeyen J.R. Sealy J. Clements A. Bhat S. Munir M. Bryant J.E. Iqbal M. The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus Viruses 2018 10 81 10.3390/v10020081 29438322 PMC5850388

298. Zou Z. Huang K. Wei Y. Chen H. Liu Z. Jin M. Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck Tembusu virus infection Sci. Rep. 2017 7 1478 10.1038/s41598-017-01554-1 28469192 PMC5431151

299. Tang Y.D. Liu J.T. Wang T.Y. Sun M.X. Tian Z.J. Cai X.H. Comparison of Pathogenicity-Related Genes in the Current Pseudorabies Virus Outbreak in China Sci. Rep. 2017 7 7783 10.1038/s41598-017-08269-3 28798304 PMC5552686

300. Xu A. Qin C. Lang Y. Wang M. Lin M. Li C. Zhang R. Tang J. A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system Biotechnol. Lett. 2015 37 1265 1272 10.1007/s10529-015-1796-2 25724716

301. Liang X. Sun L. Yu T. Pan Y. Wang D. Hu X. Fu Z. He Q. Cao G. A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus Sci. Rep. 2016 6 19176 10.1038/srep19176 26777545 PMC4726036

302. Tang Y.D. Liu J.T. Wang T.Y. An T.Q. Sun M.X. Wang S.J. Fang Q.Q. Hou L.L. Tian Z.J. Cai X.H. Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system Virus Res. 2016 225 33 39 10.1016/j.virusres.2016.09.004 27619840

303. Atasoy M.O. Rohaim M.A. Munir M. Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre-Lox System for Construction of a Stable Vaccine Vector Vaccines 2019 7 207 10.3390/vaccines7040207 31817447 PMC6963826

304. Gong Y. Chen T. Feng N. Meng X. Sun W. Wang T. Zhao Y. Yang S. Song X. Li W. A highly efficient recombinant canarypox virus-based vaccine against canine distemper virus constructed using the CRISPR/Cas9 gene editing method Vet. Microbiol. 2020 251 108920 10.1016/j.vetmic.2020.108920 33197867

305. Laudermilch E. Chandran K. MAVERICC: Marker-free Vaccinia Virus Engineering of Recombinants through in vitro CRISPR/Cas9 Cleavage J. Mol. Biol. 2021 433 166896 10.1016/j.jmb.2021.166896 33639215 PMC8574990

306. Zhang Y. Li W. Tang N. Moffat K. Nair V. Yao Y. Targeted Deletion of Glycoprotein B Gene by CRISPR/Cas9 Nuclease Inhibits Gallid herpesvirus Type 3 in Dually Infected Marek’s Disease Virus-Transformed Lymphoblastoid Cell Line MSB-1 J. Virol. 2022 96 e0202721 10.1128/jvi.02027-21 35107377 PMC8941929

307. Zhu J. Ananthaswamy N. Jain S. Batra H. Tang W.C. Lewry D.A. Richards M.L. David S.A. Kilgore P.B. Sha J. A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering bioRxiv 2021 10.1101/2021.01.19.427310 34516878 PMC8442874

308. Fokine A. Chipman P.R. Leiman P.G. Mesyanzhinov V.V. Rao V.B. Rossmann M.G. Molecular architecture of the prolate head of bacteriophage T4 Proc. Natl. Acad. Sci. USA 2004 101 6003 6008 10.1073/pnas.0400444101 15071181 PMC395913

309. Lin C. Li H. Hao M. Xiong D. Luo Y. Huang C. Yuan Q. Zhang J. Xia N. Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells Sci. Rep. 2016 6 34531 10.1038/srep34531 27713537 PMC5054376

310. Limsirichai P. Gaj T. Schaffer D.V. CRISPR-mediated Activation of Latent HIV-1 Expression Mol. Ther. 2016 24 499 507 10.1038/mt.2015.213 26607397 PMC4786916

311. Abbott T.R. Dhamdhere G. Liu Y. Lin X. Goudy L. Zeng L. Chemparathy A. Chmura S. Heaton N.S. Debs R. Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza Cell 2020 181 865 876.e12 10.1016/j.cell.2020.04.020 32353252 PMC7189862

312. Freije C.A. Myhrvold C. Boehm C.K. Lin A.E. Welch N.L. Carter A. Metsky H.C. Luo C.Y. Abudayyeh O.O. Gootenberg J.S. Programmable Inhibition and Detection of RNA Viruses Using Cas13 Mol. Cell 2019 76 826 837.e11 10.1016/j.molcel.2019.09.013 31607545 PMC7422627

313. Strutt S.C. Torrez R.M. Kaya E. Negrete O.A. Doudna J.A. RNA-dependent RNA targeting by CRISPR-Cas9 eLife 2018 7 e32724 10.7554/eLife.32724 29303478 PMC5796797

314. Johnson M.J. Laoharawee K. Lahr W.S. Webber B.R. Moriarity B.S. Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease Sci. Rep. 2018 8 12144 10.1038/s41598-018-30358-0 30108345 PMC6092381

315. Laoharawee K. Johnson M.J. Lahr W.S. Peterson J.J. Webber B.R. Moriarity B.S. Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 J. Vis. Exp. 2020 e61855 10.3791/61855 PMC7851834 33226023

316. Cheong T.C. Compagno M. Chiarle R. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system Nat. Commun. 2016 7 10934 10.1038/ncomms10934 26956543 PMC4786874

317. Lenden Hasse H. Lescale C. Bianchi J.J. Yu W. Bedora-Faure M. Deriano L. Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination J. Immunol. Methods 2017 451 71 77 10.1016/j.jim.2017.08.007 28882611 PMC5714433

318. Wu C.M. Roth T.L. Baglaenko Y. Ferri D.M. Brauer P. Zuniga-Pflucker J.C. Rosbe K.W. Wither J.E. Marson A. Allen C.D.C. Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins J. Immunol. Methods 2018 457 33 40 10.1016/j.jim.2018.03.009 29614266 PMC6124898

319. Nahmad A.D. Raviv Y. Horovitz-Fried M. Sofer I. Akriv T. Nataf D. Dotan I. Carmi Y. Burstein D. Wine Y. Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion Nat. Commun. 2020 11 5851 10.1038/s41467-020-19649-1 33203857 PMC7673991

320. Huang D. Tran J.T. Olson A. Vollbrecht T. Tenuta M. Guryleva M.V. Fuller R.P. Schiffner T. Abadejos J.R. Couvrette L. Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells Nat. Commun. 2020 11 5850 10.1038/s41467-020-19650-8 33203876 PMC7673113

321. Hartweger H. McGuire A.T. Horning M. Taylor J.J. Dosenovic P. Yost D. Gazumyan A. Seaman M.S. Stamatatos L. Jankovic M. HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells J. Exp. Med. 2019 216 1301 1310 10.1084/jem.20190287 30975893 PMC6547862

322. Voss J.E. Gonzalez-Martin A. Andrabi R. Fuller R.P. Murrell B. McCoy L.E. Porter K. Huang D. Li W. Sok D. Reprogramming the antigen specificity of B cells using genome-editing technologies eLife 2019 8 e42995 10.7554/eLife.42995 30648968 PMC6355199

323. Jaijyan D.K. Selariu A. Cruz-Cosme R. Tong M. Yang S. Stefa A. Kekich D. Sadoshima J. Herbig U. Tang Q. New intranasal and injectable gene therapy for healthy life extension Proc. Natl. Acad. Sci. USA 2022 119 e2121499119 10.1073/pnas.2121499119 35537048 PMC9171804

324. Liu J. Jaijyan D.K. Chen Y. Feng C. Yang S. Xu Z. Zhan N. Hong C. Li S. Cheng T. Cytomegalovirus-vectored COVID-19 vaccines elicit neutralizing antibodies against the SARS-CoV-2 Omicron variant (BA.2) in mice Microbiol. Spectr. 2023 11 e0246323 10.1128/spectrum.02463-23 37971259 PMC10883801

325. Zeng J. Jaijyan D.K. Yang S. Pei S. Tang Q. Zhu H. Exploring the Potential of Cytomegalovirus-Based Vectors: A Review Viruses 2023 15 2043 10.3390/v15102043 37896820 PMC10612100

326. Naeem M. Alkhodairy H.F. Ashraf I. Khalil A.B. CRISPR/Cas System Toward the Development of Next-Generation Recombinant Vaccines: Current Scenario and Future Prospects Arab. J. Sci. Eng. 2023 48 1 11 10.1007/s13369-022-07266-7 36185592 PMC9510529

327. Kimberland M.L. Hou W. Alfonso-Pecchio A. Wilson S. Rao Y. Zhang S. Lu Q. Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments J. Biotechnol. 2018 284 91 101 10.1016/j.jbiotec.2018.08.007 30142414

328. Wang X. Wang Y. Wu X. Wang J. Wang Y. Qiu Z. Chang T. Huang H. Lin R.J. Yee J.K. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors Nat. Biotechnol. 2015 33 175 178 10.1038/nbt.3127 25599175

329. Schumann K. Lin S. Boyer E. Simeonov D.R. Subramaniam M. Gate R.E. Haliburton G.E. Ye C.J. Bluestone J.A. Doudna J.A. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins Proc. Natl. Acad. Sci. USA 2015 112 10437 10442 10.1073/pnas.1512503112 26216948 PMC4547290