Current landscape of gene-editing technology in biomedicine: Applications, advantages, challenges, and perspectives.

INTRODUCTION

An enormous amount of genetic information is hidden in the nucleotide sequence; sometimes, only a single base mutation has the potential to cause incurable diseases and even death. Over the past decade, the rapid expansion of gene‐editing technology has constantly reshaped our conception of human genetics. These exquisite molecular tools have driven a massive revolution in many industries, especially the therapeutic industry. The “genomic scalpel” offers a profound opportunity to investigate genetic information, expanding our understanding of gene functions. Continued efforts to understand and elucidate these disease‐causing mutations will be crucial to improving health care for patients with genetic disorders. Previous preclinical studies have demonstrated that the pathological process can be reversed using gene‐editing molecules to inactivate or correct morbid mutations. These promising results inspired the efforts to develop gene‐editing therapeutics to treat human genetic diseases in the clinic. In addition to genetic disorders, the scope of gene‐editing applications has been extended to other conditions, including cancer and viral infections. Scientists in both biomedical research and commercial exploitation have displayed great enthusiasm for therapeutic gene editing. However, to reach the ultimate goal in which all genetic disorders can be cured by personalized gene editing, many difficult problems still need to be addressed. For instance, clinical applications require safer and more accurate engineered enzymes and more efficient delivery approaches. Regulating this technology to avoid abuse is also a focus of public concern.

In this review, we outline the development of gene‐editing technology and introduce the most commonly used gene‐editing tools. Next, we describe the current state of genome editing applications in fundamental research, focusing on developing disease models and diagnostic techniques. Finally, we summarize the clinical application of therapeutic gene editing and highlight the opportunities and challenges.

CONSTANTLY UPDATED GENOME‐EDITING TECHNOLOGY

In the 1980s, restriction endonuclease from bacteria was used to cleave the DNA double helix. 1 Since then, scientists have made a great effort to enrich the genome‐editing arsenal to manipulate gene sequences at specific loci. Different classes of genome manipulation tools are currently exploited for multiple applications in laboratory investigation, agriculture, and medicine. Common tools include zinc finger nucleases (ZFNs), transcription activator‐like (TAL) effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR‐associated (Cas) protein, base editors, and primer editors. 2 It is clear that every gene‐editing method has different mechanisms and operational constraints. 3 However, most genetic modifications from various tools (when not extended to base and primer editors) rely on generating a double‐strand break (DSB), which will trigger the endogenous repair mechanism in eukaryotic cells. 4 , 5 Two endogenous DNA repair pathways within mammalian cells are generally responsible for repairing DSBs, including nonhomologous end‐joining (NHEJ)‐mediated repair 6 and homology‐directed repair (HDR; Figure 1 ). 7 , 8 Theoretically, any gene segment can be incorporated into the genome by providing a homologous template via the HDR pathway. Given that NHEJ is typically dominant in most mammalian cells, improving the efficiency of HDR is crucial to maximizing the power of gene‐editing platforms. 9 , 10 Therefore, knowing the mechanism and characteristics of diverse genome‐editing technologies, along with choosing suitable gene‐editing agents, contributes to reaching our aspiration of performing genetic change precisely (various gene‐editing technologies have been summarized in Table 1 ).

CRISPR‐Cas nucleases: A robust and versatile genome‐editing tool

In nature, the various CRISPR‐Cas systems discovered in bacteria and archaea are adaptive immune mechanisms that silence foreign nucleic acids to resist invasion by the pathogen. 11 , 12 , 13 In brief, once Cas effectors assemble with guide RNA molecules containing spacers, the complex can bind and cleave specific sequences near the protospacer adjacent motif (PAM). 11 , 14 This mechanism inspired researchers to reprogram the DNA‐recognizing capability of the CRISPR‐Cas system by using different nucleases and guide RNA. Generally, CRISPR‐Cas systems are classified into two groups (six types and more than 20 subtypes) according to their components and mechanisms. 15 For instance, class 1 CRISPR‐Cas systems, including Type I, Type III, and Type IV, can cleave nucleotide sequences depending on multiprotein complexes. 16 In contrast, class 2 systems, which are subdivided into Type II, Type V, and Type VI, use single Cas effectors to achieve DNA cleavage. 17 , 18 Because of their unique programmability and structural advantage, class 2 systems have been widely used in multiple genome‐editing applications. Among the class 2 CRISPR‐Cas systems, Cas9, Cas12, and Cas13 are currently the research focus.

CRISPR‐Cas9 system

The natural CRISPR‐Cas9 systems are Type‐II CRISPR systems and are composed of DNA endonucleases and two RNA modules. 19 A blunt DSB could be generated by this ribonucleoprotein (RNP) complex (Figure 2 ): CRISPR RNAs (crRNAs) paired with trans‐activating crRNAs (tracrRNAs) guide nucleases to target specific loci and facilitate complex formation; then, endonucleases are responsible for cutting nucleic acids. 13 To optimize their operability, the crRNA and tracrRNA were integrated into a single guide RNA (sgRNA).

The characteristics of various Cas9 systems, including the PAM specificity, 20 spacer length, endonuclease activity, and guide RNA architecture, directly determine the precise recognition and cleavage process. 21 For example, CRISPR Cas9 from Streptococcus pyogenes (SpCas9) was the first reported Cas9 variant that was tested for manipulating genetic sequences in vitro and in mammalian cells. 22 , 23 For SpCas9 to perform normal functions, it is required for the user to choose an appropriate target location adjacent to the PAM 5′‐NGG (N represents any nucleotide) and design a 20 nt spacer within the sgRNA or crRNA/tracrRNA pair as well as an active nuclease. 22 , 24 In practice, scientists have discovered and developed many Cas9 variants to expand the scope of gene‐editing applications. 25 , 26 , 27 , 28 , 29 These Cas9 orthologs have shown diverse characteristics from SpCas9, such as smaller protein size ( Staphylococcus aureus Cas9 contains 1053 amino acids, Campylobacter jejuni Cas9 contains 984 amino acids) 30 , 31 , 32 and abundant PAM sequences that broaden the target scope ( S. thermophilus Cas9 recognizes the PAM 5ʹ‐NNAGAAW, and W represents A or T). 33 , 34

CRISPR‐Cas12 nucleases

CRISPR‐Cas12 effectors from the Type‐V CRISPR system are also RNA‐guided endonucleases. Several innate features distinguish Cas12 from CRISPR‐Cas9 systems (Figure 2 ). 18 , 35 For instance, the recognition site of most Cas12 systems is located downstream of the PAM sequence, and the cleavage site within the protospacer is farther from the PAM. In addition, many Cas12 variants usually produce a staggered nucleic acid incision with a 4–5 nt overhang downstream of the PAM sequence by using a single crRNA. 36 The first engineered Cas12 nuclease named Cas12a (formerly Cpf1) has been applied to modify the genome information in human cells. 36 , 37 The Cas12a effector inherently possesses the capacity to generate self‐required crRNA from crRNA arrays without needing tracrRNA. 38 This unique advantage means that this system has the potential to simplify multiplexed gene editing since scientists simply need to design multiple crRNA arrays to achieve this goal. 38 , 39 Within Cas12a family proteins, two orthologs from Acidaminococcus spp. (AsCpf1) and Lachnospiraceae spp. (LbCpf1) have been shown to display gene‐editing activity in human cells by recognizing the T‐rich PAM (5′‐TTTV). 40 , 41 In addition to targeting and cutting DNA, some Cas12 variants (such as Cas12 g) can cleave the RNA when guided by a single crRNA. 42 , 43 The discovery of Cas12‐family endonucleases has propelled technical innovation in genome editing.

CRISPR‐Cas13

After a series of bioinformatic analyses and computational efforts using microbial metagenomic database, some CRISPR‐Cas13 variants were identified by Feng Zhang and colleagues, including Cas13a (formerly named C2c2), Cas13b, Cas13c, and Cas13d. 44 , 45 Similar to Cas9 or Cas12 systems, Cas13 systems possess programmable nuclease activity and have the potential to be developed as RNA editing tools. The Cas13a effectors from the class 2 Type VI system are RNA‐guided RNA endonucleases that have been optimized and show activity in manipulating RNA (Figure 2 ). 46 Recently, specific orthologs of Cas13, including Cas13b and Cas13d, were leveraged for RNA knockdown and manipulation and exhibited stability and high efficiency in RNA editing. 47 , 48 In addition to RNA knockdown and editing, Cas13 systems have been reformed for nucleic acid detection. 49 The Cas13 protein will cleave any single‐stranded RNA in its vicinity after binding and cutting the targeted sequence. 50 Based on the collateral cleavage activity of Cas13‐family endonucleases, Zhang et al. established a diagnostic platform termed specific high‐sensitivity enzymatic reporter unlocking (SHERLOCK). 51 , 52 Compared with the traditional nucleic acid detection method quantitative polymerase chain reaction (qPCR), this diagnostic technology can detect RNA or DNA within 10–15 min with great sensitivity. 53 At present, more Cas13 orthologs have been discovered and tested for other applications, such as transcriptome regulation and RNA visualization in live cells. 54

Base editors: The pearl in the crown of gene editing

Modest gene mutations, even a single nucleotide variation, may trigger the occurrence of genetic disease in humans. 55 Currently, many genome‐editing tools have been applied to correct genetic mutations. Of these cases, most strategies must generate DSBs and depend on the HDR pathway. However, common single base modification, which depends on the DNA repair pathway induced by DSBs, is far from satisfactory. 56 , 57

To solve these issues, two different types of base editors based on CRISPR‐Cas have been developed for precise base manipulation. One is cytosine base editors (CBEs), and the other is adenine base editors (ABEs). 58 These tools were able to install point mutations without DSB through the single‐strand repair pathway (Figure 1 ). 59

CBEs install C•G‐to‐T•A mutations

The basic components of CBEs include a personalized sgRNA and a multifunctional fusion protein that consists of catalytically deficient CRISPR‐Cas nuclease and single‐stranded DNA cytidine deaminase (Figure 2 ). 59 According to guidance by sgRNA, Cas effectors can bind the target sequence and create an ssDNA R‐loop without generating DSBs. Generally, the editing window is a short fragment of exposed ssDNA (positions 4–8 are located in the protospacer if positions 21–23 are PAM). The cytidine deaminases are able to complete the transition point mutations (cytosines are converted to uracils) on the noncomplementary strand. 60

To date, scientists have created multiple versions of CBEs to gain greater efficiency and safety. The first‐generation base editor, termed BE1, was formed using the cytidine deaminase enzyme APOBEC1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐1) from Rattus norvegicus and a dead Cas9 (dCas9) without endonuclease activity. 61 However, the mismatched base pair (U•G) is rectified through the base excision repair pathway, which may result in poor BE1 editing effectiveness in mammalian cells. 62 , 63 To boost the base editing efficiency, the second‐generation base editor BE2 was developed by adding an extra uracil glycosylase inhibitor (UGI). 61 , 64 Because of the UGI, the U•G intermediate could remain in vivo longer to obtain stable base editing outcomes. 65 To further increase the editing efficiency, the third version of the base editor (BE3, also described as CBE) replaced dCas9 with Cas9 nickase (Cas9n). 59 Once the nondeaminated strand has been cleaved, the activated cellular repair mechanism will use the U‐containing DNA strand as a template for fixing the gap. Additionally, many variants of BE3 have been engineered to improve the editing precision by narrowing the editing window, namely, variants such as YE1‐BE3, YE2‐BE3, and YEE‐BE3. 66 , 67 Most recently, the fourth‐generation base editor BE4, which possesses two UGI proteins, was created for superior editing outcomes. 68 , 69

ABEs install A•T‐to‐G•C mutations

The initial design of ABEs was a Cas9n tethered to an adenosine deaminase (ADA). As originally conceived, ABE can deaminate adenine within the R‐loop into inosine, which was eventually restored to guanine. 58 However, the natural ADA cannot convert adenosine within ssDNA to inosine. Therefore, researchers have tried to reform the tRNA‐specific ADA (ecTadA) from Escherichia coli using directed evolution. After complex protein engineering, the desired mutation was discovered and fused to Cas9n. The final architecture of ABEs was composed of a heterodimeric ADA (ecTadA‐Tad*) and a Cas9n. This version was named ABE7.10, and it is the most commonly used base‐editing tool. 58 The discovery of ABE7.10 offers the possibility of extending the range of base editing. Similar to CBEs, ABEs have been continuously upgraded to pursue superior editing effectiveness. For instance, ABEmax with several nuclear localization signal peptides has been developed based on ABE7.10. 68

Prime editors: A potent new base modification technology

Four transition point mutations (A→G, T→C, C→T, and G→A) have been implemented by using CBEs and ABEs without reliance on the HDR pathway. However, the implementation of base transversions and the other eight types of point mutations (A→C, A→T, T→A, T→G, C→A, C→G, G→C, and G→T) require novel technology. In 2019, Liu's lab reported on a pioneering gene‐editing tool called prime editor, which could not only install all possible types of DNA point mutations but could also induce small deletions or insertions without donor DNA templates. 70 The current prime editor consists of a prime editing guide RNA (pegRNA) and a Cas9n fused to engineered reverse transcriptase (Figure 2 ). The protein complex guided by pegRNA nicks the target DNA strand and mediates subsequent reverse transcriptional reactions. In fact, the pegRNA is a modified sgRNA that has an extra RNA sequence at the 3′ end. 70 This guide RNA containing the desired gene fragment is responsible for recognizing the target sequence and serves as a template for subsequent reverse transcription. In brief, the DNA single‐strand nicked by Cas9n could use the pegRNA as the template to execute reverse transcription. After the reaction, the mutations are placed at the target site, generating a 5′ DNA flap and a 3′ DNA flap containing the edited sequence. Last, the 5′ DNA flaps were generally removed by the cellular DNA repair pathway. 71 Compared with other genome‐editing tools, prime editors have advanced in some respects, including a more extensive application range, as well as better editing accuracy and safety with few byproducts. 60 The superprecise CRISPR tool enhances our ability to manipulate genetic information and has the potential to promote the development of gene therapy. 72

ZFNs: The first generation of mature gene‐editing technology

ZFNs, which consist of zinc finger motifs and the FokI restriction endonuclease, are considered one of the earliest gene‐editing tools (Figure 2 ). Zinc finger proteins are responsible for recognizing and binding nucleotide sequences and are versatile transcription factors in eukaryotic organisms. 73 Each zinc finger protein consists of approximately 30 amino acids and forms a ββα structure, with a Cys2‐His2 domain interacting with the zinc atom. 74 According to the three‐dimensional cocrystal structure, the NDA recognition capacity of zinc finger proteins (ZFs) relies on the α‐helical reading head bound with 3 bp within the major groove of the double helix. 75 The Type II restriction enzyme FokI derived from Flavobacterium okeanokoites requires dimerization to complete DNA cleavage, and the cutting ability is independent without side effects. 76 , 77 Generally, dimerized ZFNs can recognize 18–36 bps of the target sequence and generate a staggered DSB with a 5′ overhang. In principle, ZFNs can target any desired gene sequence by using various zinc finger proteins. In addition, the DNA binding specificity also depends on the number of fingers and the target location. However, the production of highly specific ZFNs is far from satisfactory since the assembly and optimization procedure required a great deal of time and effort. To extend the range of DNA recognition, many strategies, such as “two‐finger modules 78 ” and the “open system,” 79 have been established to reform and optimize the structure and function of ZFNs. For example, scientists obtained new ZFN architectures with a larger recognition range by using new linkers derived from a cleavage‐based bacterial selection system. 80

Many advantages, including low immunogenicity and appropriate gene size, have prompted the widespread use of ZFNs as gene‐editing tools for multiple applications. Unfortunately, the further large‐scale application and popularization of ZFNs were hindered by some technical barriers. Admittedly, the synthesis and assembly of zinc finger proteins are time‐consuming and require technical expertise. In addition, the related technology patents belong to several agencies, which hinders the widespread use of ZFNs.

TALENs: A flexible protein‐based editing system

Because of the shortage of publicly available resources, it is difficult to construct ZFNs. To overcome these technical limitations, a protein‐based DNA editing platform known as TALENs was established. TALENs contain a catalytic FokI endonuclease fused to DNA‐binding domain TALEs (Figure 2 ). Natural TAL effectors have been discovered in plant pathogenic Xanthomonas bacteria, and they change the transcription in the host cells. 81 The DNA targeting capacity of TALEs depends on highly conserved tandem arrays, which consist of 10–30 repeats. Individual TALE repeats that contain approximately 33–35 amino acids could target a single nucleotide accurately. The architecture of a single TALE includes two helix bundles and a loop. Notably, two hypervariable residues (residues 12 and 13) within the loop structure, which is currently named the repeat variable di‐residue (RVD), determine the specificity and affinity of each effector. 82 The RVD can not only ensure the stability of the loop structure but can also target specific nucleotides. 83 To date, four identified RVDs, HD (HD represents the residules of histidine and aspartic acid, specifies cytosine), NG (NG represents the asparagine and glycine residues, specifies thymine), NI (NG represents the asparagine and isoleucine residues, specifies adenine), and NN (NN means two asparagine residues, specifies guanine or adenine), have been widely employed to generate the desired TALENs for multiple gene‐editing applications.

Similar to ZFNs, the platform based on TALENs can be reprogrammed to bind arbitrary sequences by rearranging TALE repeats, but the complicated assembly procedure limits its promotion. To accelerate the assembly process, several strategies have been implemented, including the Golden Gate cloning system, restriction enzyme and ligation technology, fast ligation‐based automatable solid‐phase high‐throughput system, and ligation‐independent cloning technology. 84 In addition to their programmability, TALENs have several obvious advantages: simplified design and construction and a flexible range of recognition. For instance, dimerized TALENs are usually engineered to bind 36‐bp sequences or even longer. Of course, there are some disadvantages that must be solved. The large size of TALENs (approximately 3 kilobases [kb]) may cause delivery difficulties. During the delivery of TALENs, the repeats are susceptible to rearrangements, 85 which carries a risk of genetic manipulation.

GENOME EDITING FOR FUNDAMENTAL RESEARCH: DISEASE MODELLING AND DISEASE DIAGNOSIS

Targeted genome editing tools for disease modeling

The unprecedented ability to create a gene modification at a specific site has driven the development and upgradation of medical disease models. Numerous successful disease models have been established for further detailed pathologic studies.

Cardiovascular disease (CVD) models

CVD is considered a critical public health issue because of its high mortality and morbidity. 86 Most CVDs are inherited diseases characterized by monogenic mutations or a complex of heterozygous mutations. 87 Carroll et al. created a Cas9 transgenic mouse model that only expresses a high level of Cas9 in the heart, but no evident burden was observed in vivo. 88 Driven by adeno‐associated virus (AAV), sgRNA was delivered to the Myh4 locus to induce an accurate and rapid depletion of cardio‐relevant genes and subsequently show severe cardiomyopathy. The fibrillin‐1 encoded by the FBN1 gene is the fundamental component of the connective tissue matrix. Heterozygous mutations in FBN1 often correlate with the phenotype of Marfan syndrome (MFS). 89 , 90 Using pigs as a human biomedicine model could promote the study of human diseases. Many medical studies require a suitable animal model that resembles humans to repeat experiments. For example, the porcine model is considered a more suitable candidate for investigating CVD because of its unique biological characteristics. Specifically, the anatomical, physiological, and genetic characteristics of pigs are similar to those of humans. 91 Some porcine somatic cells have been modified by ZFNs to generate a cloned pig with a heterozygous FBN1 mutant (+/Glu433AsnfsX98), which exhibited phenotypes (scoliosis, pectus excavatum, and structural damage in the aortic medial tissue) similar to those of patients with MFS. 92 Based on a similar strategy, Yang et al. took advantage of ZFNs and electroporation to knock out the gene encoding peroxisome proliferator‐activated receptor‐gamma ( Ppar‐γ ) in primary porcine cells and then generated an invaluable Ppar‐γ knockout (KO) porcine model for studying CVD. 93

Metabolic disease models

Metabolic disorders represent a dysfunctional state in the body, hampering the shifting of food energy into cell bioreactions. 94 Leptin ( Lep) and its corresponding receptor ( LepR ) are responsible for inhibiting fat synthesis and balancing energy metabolism. 95 To elucidate their pivotal function in glycolipid metabolism, scientists have created multiple animal models, which are typically represented by obesity mutant mouse models and diabetes mutant mouse models. 96 Using CRISPR‐Cas9 technology, Bao and colleagues established LepR knockout rats, which ultimately exhibited complications of obesity and diabetes. 97 Furthermore, Chen et al. successfully installed diverse LepR mutations into mouse embryos by TALENs, generating several transgenic rats. Obesity, insulin resistance, and metabolic disorders were observed in some rats with a frame‐shifted or premature stop codon mutation. The study reported the first rat models for obesity research based on the Sprague Dawley strain. 98 As the key regulator in the metabolism of glucose and lipids, cAMP (cyclic adenosine monophosphate)‐responsive element‐binding protein 3‐like 3 (CREB3L3) is predominantly expressed in the liver and intestine. 99 To investigate the different functions of CREB3L3 within various tissues, some Japanese scholars invented liver‐ and small intestine‐specific CREB3L3 knockout mice through the CRISPR‐Cas9 system. With the help of these mouse models, these investigators provided a new understanding regarding the role of CREB3L3 in different metabolic pathways. 100

Models for neurodegenerative diseases (NDs)

NDs (such as Huntington's disease [HD], Alzheimer's disease [AD] and Parkinson's disease [PD]) have always been a threat to human health. Due to the deficiency in effective diagnostic and therapeutic schemes, the public health care system suffers from stress. 101 In particular, the dearth of suitable disease models has hampered progress in the field. Currently, many in vitro and in vivo studies have demonstrated that the huntingtin ( HTT ) gene is closely related to the occurrence of HD. 102 , 103 , 104 To elucidate the pathogenesis of HD, diverse animal models have been investigated through gene editing. After introducing truncated mutant HTT into HD140Q knock‐in mice through the CRISPR‐Cas system, investigators found that exon 1 HTT is a key pathogenic factor of HD. 105 Furthermore, Yan et al. generated a gene‐engineered porcine model with full‐length mutant HTT using CRISPR‐Cas9 and somatic nuclear transfer technology. These large animal models recapitulated overt and selective neurodegeneration exhibited in HD patients, while pathological features can be germline‐transmitted to their progeny. 106

The amyloid precursor protein ( APP ) gene has also been extensively studied in NDs, specifically AD. 107 To obtain APP‐overexpressing cell lines, the partial coding regions of APP were incorporated into the genome of mouse fibroblast cells by ZFNs. These modified cells illustrate how APP mutants influence relevant signaling pathways to induce the onset of AD. 108 TALENs were also used to create A673 V and A673T variants based on human‐induced pluripotent stem cells (iPSCs), which ultimately developed various levels of AD‐featured biomarkers. 109 In addition, Paquet et al. installed both heterozygous and homozygous mutations of A PP or presenilin (PSEN) 1 in human iPSCs enabling the appearance of early AD symptoms. 110

Hereditary ocular diseases

In recent years, emerging gene‐editing technology has made it possible to establish hereditary eye disease models, which help to explain the pathological mechanisms and to identify pathogenic genes. The existing eye disease models are primarily concentrated on retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and retinoblastoma, among others.

Receptor expression enhancer protein 6 ( REEP6 ) is highly expressed in rod photoreceptor cells, contributing to the formation of the endoplasmic reticulum. 111 Several biallelic variants of REEP6 have been identified in seven unrelated individuals affected with RP. Therefore, one of the verified missense mutants, which is known as REEP6.1 , was integrated into the murine genome through CRISPR‐Cas9, inducing clinical manifestations of RP, such as retinal degeneration and the malfunction of rod photoreceptors. 112 LCA, a congenital eye disease, can lead to retinopathy and early blindness. 113 At present, numerous mouse models have been generated to support the functional exploration of all other LCA genes. For instance, some evidence has shown that KCNJ13 mutants are closely linked to the early onset of LCA. Subsequently, different concentrations of spCas9RNA and sgRNA were delivered into zygotes using microinjection to generate a KCNJ13 gene‐null mouse model. The results show that high concentration‐derived mice are more efficient and compatible with mimicking the pathogenesis of KCNJ13 ‐induced LCA and that the deficiency of KCNJ13 causes photoreceptor degeneration. 114

Other diseases

Von Willebrand disease (VWD) is the most common bleeding disorder and is characterized by coagulation disorders. 115 Hai et al. reported a convenient and efficient method for preparing VWD models. They took advantage of the CRISPR‐Cas9 platform to knock out the double allele of the VWF gene in pigs. This miniature pig model perfectly circumvents the current obstacle in which small rodents cannot highly recapitulate the disease hallmarks of human patients. 116

Rett syndrome (RTT) is a serious disorder affecting children's neurodevelopment. Arrested growth predominantly occurs in females aged 6–18 months old. However, for males, RTT is fatal during the fetal period. 117 Methyl‐CpG binding protein 2 ( MECP2 ) is an X‐linked gene, the mutations of which correlate with the rise of the autism spectrum. 117 , 118 A research team from China employed TALENs to create the first MECP2 mutation model based on rhesus and cynomolgus macaques. These models have shown almost identical embryonic lethality with human boys suffering from RTT. Additionally, when using the same gene‐editing system, this team generated another cynomolgus monkey model with mutagenesis of the RTT, which displayed stark abnormalities in physiology, behavior, and body structure. 119

Thanks to the rapid advancement of genome‐editing technologies, numerous severe and refractory diseases also have available disease models for study, such as sickle cell disease (SCD), 120 Niemann–Pick disease, 121 and Duchenne muscular dystrophy. 122 Progress in the genetic engineering area will further extend the application of animal models to elucidate the more sophisticated molecular mechanisms and raise entirely new prospects for therapeutic modalities.

New disease diagnostic tools based on the CRISPR‐Cas system

In many cases, efficient therapeutic intervention relies on the timely and accurate diagnosis of disease. The currently relevant application of gene editing has shown unique advantages in the diagnosis of diseases, especially cancer and viral infections. 123 , 124 The currently reported CRISPR‐Cas systems can be broadly divided into two classes and some subtypes, according to the nature of the effector protein complex. 15 , 18 Of these systems, the Class 2 CRISPR system, including Types II, V, and VI, is principally applied to develop novel diagnostic tools. For instance, one group made an attempt to create a CRISPR‐Cas9n‐mediated strand displacement amplification method (abbreviated as CRISDA). Multiple experimental results indicate that CRISDA has the potential to be a powerful diagnostic tool because of its single‐nucleotide specificity and versatility. 125

One interesting application of the CRISPR‐Cas system is in the diagnosis of cancer. Cancer, as an important disease threatening human health, has always had a very high mortality rate. The important reason for mortality is that patients often miss the best time for treatment because of the shortage of early diagnostic approaches. The efficient early diagnosis of cancer is critical to making an effective intervention for disease progression and providing a better quality of life. In 2017, Gootenberg et al. proposed a brand‐new concept using CRISPR‐Cas13n‐mediated diagnostic techniques, which offers another ultrasensitivity and specificity option for testing RNA and DNA sequences. 49 , 51 The platform, SHERLOCK, offers a chance to screen disease‐causing mutations. Using this method, researchers found that cancer‐related EGFR‐L858R (EGFR, epidermal growth factor receptor) and BRAF‐V600E mutations with allelic fractions as low as 0.1% can be detected in cell‐free DNA fragments. In subsequent studies, scientists successfully detected EGFR‐L858R and EGFR‐T790 M mutations in DNA fragments derived from non‐small cell lung cancer (NSCLC) patients. 126 Inspired by SHERLOCK, investigators used Cas12a and recombinase polymerase amplification to establish another diagnostic tool named DNA endonuclease‐targeted CRISPR trans reporter (DETECTR), which has attomolar sensitivity and detects infectious viruses associated with cancer. Typically, human papillomavirus (HPV) 16 and HPV18, the two most pro‐oncogenic HPV species, can be identified and distinguished by DETECTR. 127 In practical applications, the detection efficiency is satisfactory, and the detection rate fluctuates between 90% and 100%. 128 Furthermore, a reusable electrochemical biosensor consisting of the CRISPR‐Cas13a system and a catalytic hairpin DNA circuit has been leveraged to screen tumor‐related RNA for the early diagnosis of NSCLC. 129

Another interesting application of the CRISPR‐Cas system is in the diagnosis of infectious diseases. Most currently, global health care systems have been undermined to an unprecedented extent by the novel coronavirus (COVID‐19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). 130 , 131 , 132 Accurate and rapid diagnosis will benefit patients to receive timely treatment. However, enzyme‐linked immunosorbent assat (ELISA) and real‐time or reverse transcription‐polymerase chain reaction (RT‐PCR) are common and classical molecular detection tools that require intricate equipment and fastidious work processes. 133 It is imperative to establish a portable, accurate, and convenient diagnostic system for detecting infectious disease pathogens. Diagnostic CRISPR systems have outstanding superiority in terms of their ultrahigh sensitivity, portability, and specificity. For instance, the Cas12 and Cas13 nucleases have been reprogrammed to detect the nucleic acids of SARS‐CoV‐2. 134 , 135 , 136

Viruses, whether DNA viruses or RNA viruses, are important pathogens of infectious diseases. RNA viruses that have been studied abundantly in recent years include dengue, 137 Zika, 49 and a recent spotlight of interest, SARS‐CoV‐2. 138 For SARS‐CoV‐2 detection, the SHERLOCK and DETECTR detection systems mentioned above have been modified and upgraded to achieve higher efficiency in virus extraction, 139 more flexibility in sensing capability, and a more streamlined mechanical process. 140 , 141 In 2022, Lu et al. reduced the detection time of CRISPR‐based assays while ensuring accuracy through a hybrid strategy, which includes adjusting the kinetics of Cas12a and using more flexible crRNA designs. 134 In addition to RNA viruses, CRISPR/Cas‐based diagnostic methods can identify DNA viruses, such as BK virus (BKV), cytomegalovirus (CMV), and Epstein–Barr virus (EBV). 142 , 143 The researchers used the SHERLOCK system to detect BKV or CMV in serum samples and then verified the results by qPCR. The comparison results demonstrated that the two approaches have almost the same specificity. To date, the Food and Drug Administration (FDA) has authorized a CRISPR‐based COVID‐19 diagnostic tool for emergency use. The successful translational application of the CRISPR‐Cas system in COVID‐19 diagnosis indicated that this versatile technology has the potential to be applied to other severe epidemic disease diagnoses in the future.

CURRENT APPLICATION OF GENE EDITING TO THE TREATMENT OF HUMAN DISEASES

The ultimate aspiration pursued by researchers is to use gene‐editing technologies to treat human diseases without undesired side effects. The current gene‐editing techniques have provided a brilliant opportunity for precise intracellular gene manipulation, which can not only be used to induce mutations, corrections, or deletions but can also introduce foreign genes at specific sites. 144 , 145 , 146 Additionally, precise genetic manipulation can efficaciously reduce the risk of insertion mutations in some cell therapies. 147 Admittedly, novel therapies offer an attractive opportunity to save patients’ lives. In contrast, the consequence of gene editing is unknown and fraught with potential risks. Therefore, performing sufficient preclinical research is an important prerequisite for promoting gene editing for clinical application. As described in the previous section, advances in disease modeling and diagnostic techniques have driven the development of preclinical research on gene‐editing therapeutics. 148 , 149 According to these unique advantages and the promising outcomes of preclinical studies, various gene‐editing agents have been leveraged to treat human diseases in clinical trials.

Generally, the strategies of gene‐editing therapeutics contain two modes, including in vivo and ex vivo strategies (Figure 3 ). To date, numerous pre‐ and clinical studies of gene editing have been scattered across various countries. In this section, we will provide a comprehensive overview of this clinical information and primarily discuss the progress in therapeutic gene editing in clinical trials (relevant clinical trial information is summarized in Table 2 ). Additionally, we also introduce some interesting preclinical studies and highlight several current novel therapeutic strategies and concepts.

Cancer immunotherapy

There are currently more than 70 clinical trials in the NIH clinical trial database that involve gene editing‐mediated therapy, including the use of ZFNs, TALENs, or CRISPR/Cas, and nearly 50% of these trials are related to neoplasms. Cancer immunotherapy is widely considered one of the most significant advances in biological research in recent years. The development of adoptive T‐cell therapy is the most prominent. The advent of cytotoxic T lymphocyte, T‐cell receptor transgenic (TCR) T cells, chimeric antigen receptor T cells (CAR‐T) and other therapeutic products has alleviated the symptoms and prolonged the lifetimes of patients with cancer.

In the field of gene‐editing‐mediated cancer immunotherapy, as many as 19 clinical trials have focused on CAR‐T therapy, including hematological and solid tumors. It is well known that CAR‐T‐cell therapy has shown convincing evidence during clinical trials for the treatment of hematological malignancies. 150 However, as a customized therapeutic product, the widespread use of CAR‐T cells is limited. Currently, the development of universal CAR‐T (UCAR‐T) products is a major trend in the area of T‐cell therapy. 151 The emergence of UCAR‐T cells expands the range of application, improves the feasibility of application, and may also reduce the cost of treatment. Even if the program is still facing technical barriers, there is no doubt that UCAR‐T therapy will be a key development direction in the future. As a leader in UCAR‐T, the French company Cellectis has developed an allogeneic CAR‐T technology platform based on TALEN gene‐editing technology. Cellectis Inc. has invented four “off the shelf” CAR‐T products by inactivating the TCR and CD52 genes: UCART22 targets CD22 to treat B‐cell acute lymphoblastic leukemia (B‐ALL; NCT04150497 ); UCART123 targets CD123 to treat acute myeloid leukemia ( NCT03190278 ); UCARTTCS targets the CS1 antigen to treat relapsed/refractory multiple myeloma (MM; NCT04142619 ); and UCART19 is the first UCAR‐T product for treating B‐ALL ( NCT02746952 ; NCT02808442 ). ALLO‐501A, a UCAR‐T product developed by Allogene Therapeutics using a similar concept, has already initiated clinical trials for treating B‐cell lymphoma ( NCT04416984 ). Another company, CRISPR Therapeutics, has invented three UCAR‐T therapies based on the CRISPR gene‐editing system, namely, CTX110, CTX120, and CTX130. Unlike Cellectis Inc., CRISPR Therapeutics chooses to destroy β2M and TCR loci to reduce the risk of rejection. CTX110 is a CD19‐specific CAR‐T that is primarily used to treat B‐cell malignancy ( NCT04035434 ). CTX120 kills MM cells by recognizing BCMA ( NCT04244656 ), while CTX130 is used to treat CD70‐expressing T‐cell lymphoma and renal cell carcinoma ( NCT04502446 ; NCT04438083 ). In addition, some researchers designed “off the shelf” CAR‐T cells against CD19 or mesothelin to treat B‐cell hematological or solid tumors ( NCT03166878 ; NCT03545815 ).

Apart from UCAR‐T therapy, gene modification technology can be used to disrupt endogenous genes to improve the effectiveness of CAR‐T cells. For example, in a Phase I clinical trial, researchers used the CRISPR system to inactivate hematopoietic progenitor kinase 1, an intracellular negative regulator of T‐cell proliferation and signal transduction, to enhance the effect of CD19 CAR‐T cells ( NCT04037566 ). In another Phase I clinical trial conducted in the Chinese PLA General Hospital ( NCT04976218 ), CAR‐T cells that targeted EGFR were edited by CRISPR‐Cas9 to knock out transforming growth factor beta (TGF‐β), which is considered a major regulatory factor in the immunosuppressive tumor microenvironment (TME), and CAR‐EGFR‐TGFβR‐KO T cells may relieve the hostile TME in solids and improve the treatment effect. Antigen loss is the main reason for the failure of tumor immunotherapy; one of the strategies is to target more than one target simultaneously. A clinical trial conducted by the Chinese PLA General Hospital evaluated the feasibility and safety of universal bispecific CD19 + CD20 + and CD19 + CD22 + CAR‐T cells for treating relapsed or refractory B‐cell leukemia and lymphoma ( NCT03398967 ). 152

The well‐known immune checkpoints PD‐1 and CTLA‐4 can significantly inhibit the activity of T cells. Tumor cells can easily escape the immune response by virtue of this mechanism, resulting in poor therapeutic effects. Using gene‐editing tools to destroy endogenous immune checkpoint genes to enhance the antitumor effect is a novel treatment strategy that is widely used in the treatment of solid tumors. In 2016, the first clinical trial of PD‐1 knockout T cells for treating NSCLC was launched at West China Hospital of Sichuan University ( NCT02793856 ). Investigators took advantage of CRISPR‐Cas9 to edit the PD‐1 gene of T cells ex vivo. After culturing and expanding in vitro, the T cells were reinfused into the subject, which supported the safety and feasibility of this therapy in NSCLC for the first time. 153 Additionally, in a CRISPR‐related clinical trial led by Professor Carl June, researchers eliminated the genes encoding PD‐1 and endogenous TCR through CRISPR‐Cas9, effectively enhancing the effect of CAR‐T cells targeting melanoma ( NCT03399448 ). At present, PD‐1 knockout T cells have also been used for treating advanced hepatocellular carcinoma ( NCT04417764 ), invasive bladder cancer ( NCT02863913 ), metastatic renal cell carcinoma ( NCT02867332 ), esophageal cancer ( NCT03081715 ), and EBV‐associated malignancies ( NCT03044743 ), elevating the progress in cancer immunotherapy. 154 , 155 , 156 , 157

In addition to adoptive cell transfer therapy, many novel therapeutic strategies have been tested for treating tumors in preclinical studies. For example, the success of the SARS‐CoV‐2 mRNA vaccine depended on the advanced lipid nanoparticle (LNP) delivery system. 158 , 159 This efficient delivery vehicle has the potential to perform therapeutic gene editing in vivo. In a preclinical study, novel amino‐ionizable LNPs encapsulating Cas9 mRNA and gRNAs were injected into orthotopic glioblastoma to disrupt the polo‐like kinase 1 gene. In animal models, this strategy had positive and safe therapeutic outcomes. 160 Gao et al. constructed a new Cas13a expression vector containing a nuclear factor κB (NF‐κB)‐specific promoter and U6 promoter. The expression of Cas13a is controlled by NF‐κB, which is widely overactivated in various cancers. Once the promoter is activated, the expression of endogenous oncogenes can be regulated by designing different sgRNAs within the vector. 161 Furthermore, prime editors and some Cas variants circumvent specific adverse effects in traditional genome editing and catalyze the editing process without the requirement for DSBs. 162

Infectious diseases caused by viruses

Genome editing is expected to become a powerful tool in antiviral therapy that acts by modifying infection‐related genes required for viral invasion and replication in host cells. Through gene editing, virus‐resistant immune or stem/progenitor cells can be produced, which could prevent or alleviate viral diseases. 163

C‐C chemokine receptor type 5 (CCR5) and C‐X‐C chemokine receptor type 4 (CXCR4), the main auxiliary receptors for human immunodeficiency virus (HIV), play major roles in the initial infection and the establishment of stable infection, respectively. Additionally, homozygous carriers of the CCR5 Delta 32 mutation are naturally resistant to HIV infection, suggesting that the artificial deletion of CCR5 may be used to endow T cells with the feature of HIV infection resistance. 164 Previous studies have used different genome editing tools to inactivate the CCR5 gene and CXCR4 gene on CD4+ T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs) successfully, 165 , 166 , 167 and these findings showed that these cells are not susceptible to HIV. 168 , 169 Among anti‐HIV therapies, the strategy of deleting the CCR5 gene through ZFN is relatively mature, and many clinical trials have been approved and undertaken. The first clinical trial using gene editing to treat HIV was led by Carl June in 2009. Researchers used ZFNs (SB‐728) to inactivate the CCR5 gene of autologous CD4+ T cells and reinfused these genetically modified T cells (SB‐728‐T) into 12 recruited patients. The results showed that except for one patient who exhibited serious adverse transfusion reactions, the remaining patients were tolerant to genetically engineered T cells, 170 indicating that CCR5‐modified autologous T‐cell infusion is safe and feasible ( NCT00842634 ). Subsequently, a plurality of clinical trials focused on determining the therapeutic dose and impact of SB‐728‐T to establish an efficacious clinical protocol ( NCT03666871 , NCT01044654 , and NCT01252641 ). With the continued emphasis on the safety and effectiveness of immunotherapy, novel treatment strategies have emerged, including electrotransfecting CCR5‐specific ZFN mRNA to edit T cells ( NCT02225665 , NCT02388594 ) or knocking out the CCR5 gene in CAR‐T cells to treat HIV ( NCT03617198 ). Scientists wished to explore the effects of genetically modified T cells on HIV infection resistance under these strategies. In addition to genetically modifying T cells, scientists would like to modify hematopoietic stem or progenitor cells to create a cell pool in vivo that can continue to produce T cells resistant to HIV infection. In a clinical study, researchers from the City of Hope Medical Center delivered CCR5‐specific ZFN mRNA into HSPCs to evaluate its safety in patients infected with HIV‐1 ( NCT02500849 ). 171 Moreover, in a recent clinical study, Chinese scientists designed a stable CRISPR‐Cas9 system to edit the CCR5 gene of donor‐derived CD34 + hematopoietic stem cells (HSCs) and infused these cells back into the patient. 102 They then evaluated the feasibility and safety of this therapeutic strategy in patients infected with HIV. After patients with ALL and HIV infections received the infusion of these autologous CCR5‐deficient HSPCs, the acute lymphocytic leukemia was in complete remission, and CCR5‐deficient donor cells remained in the body for more than 19 months. This case indicated that in humans, CCR5‐inactivated HSPCs contributed to long‐term hematopoietic system reconstruction. 172 However, the low efficiency of gene editing limits the therapeutic effect, suggesting that the focus of this research may be to improve the editing efficiency. The above cases have supported the immense potential of gene‐editing technology in treating HIV and put forward further requirements for the efficiency of gene editing and the optimization of the treatment scheme.

Another strategy for antiviral therapy based on gene manipulation is to target the viral genome associated with viral replication and assembly directly. In addition to addressing HIV infection, gene‐editing platforms have been applied to other causative agents, including HPV, hepatitis B virus (HBV), and EBV. The main etiological factors of cervical cancer are some specific oncoproteins. The HPV E6/E7 genes encode important oncoproteins related to the neoplastic transformation of the disease. Multiple clinical trials have attempted to introduce different gene‐editing modules to target and destroy the DNA of HPV16/18 E6 or E7 directly in vivo. The researchers hope that the tumorigenic process will be reversed in situ and that the incidence of cervical cancer will be reduced ( NCT03226470 , NCT03057912 , and NCT02800369 ). 173 , 174 , 175 A similar idea was adopted for the elimination of HBV. To inhibit HBV replication, researchers used lentivirus to deliver CRISPR‐Cas9 nuclease and HBV‐specific sgRNA into HepG2 cells bound to HBV. 176

The COVID‐19 led the World Health Organization to declare a pandemic in March 2020, and it remains a significant threat to human health worldwide. Zhang et al. designed a novel diagnostic tool termed SHERLOCK to detect the nucleic acids of viruses. 126 Inspired by the SHERLOCK system, American scientists transformed Cas13a into an antiviral agent that was programmed to detect and destroy RNA viruses in human cells in another novel study. The antiviral activity of Cas13a and its diagnostic ability were combined to construct a system that may be used to diagnose and treat viral infections, which is termed Carver (i.e., Cas13‐assisted restriction of viral expression and readout). 177

Hematological diseases

Many hematological disorders are caused by genetic mutations, including thalassemia, hemophilia, and SCD. The correction of erroneous gene mutations can be accomplished by gene‐editing technology, which undoubtedly holds promise for treating hereditary hematological diseases.

The HBB gene mutation on chromosome 11 reduces β globin chain production, which in turn causes β‐thalassemia. 178 HSCs have significant advantages in reconstructing or restoring human hematopoietic function. A clinical experiment attempted to modify induced HSCs from patients to correct the mutated HBB gene and then transfuse them back into the patients to restore their normal hemoglobin production ability ( NCT03728322 ). The presence of fetal hemoglobin (HbF) can in some cases alleviate the symptoms of thalassemia, but HbF production is suppressed by BCL11A. 179 , 180 Thus, knocking this gene out or down leads to a suitable approach to treating SCD and beta‐thalassemia. An experimental CRISPR‐based therapy (known as CTX001) invented by CRISPR Therapeutics has undergone two clinical trials for treating SCD and thalassemia ( NCT03655678 ; NCT03745287 ). The key strategy underlying CTX001 is to use CRISPR‐Cas9 to modify the BCL11A gene within CD34 + human hematopoietic stem and progenitor cells to increase the production of HbF in vivo. Sangamo Therapeutics and Sanofi also conducted two clinical trials using a similar strategy. However, ZFNs were used instead of a CRISPR/Cas system for genetic modification ( NCT03432364 , NCT03653247 ). Furthermore, the appearance of base editors that have the capacity to manipulate a single base without DSBs provides an attractive option to cure sickle‐cell disease. Liu et al. generated a bespoke base editor to convert the pathogenic gene ( HBB S , hemoglobin subunit beta allele) into a nonpathogenic gene ( HBB G , Makassar β‐globin). Based on this strategy, approximately 80% of hematopoietic stem and progenitor cells derived from patients could be engineered in vitro. Furthermore, humanized SCD mice that received edited HSPCs exhibited reduced splenic and hematologic pathology, compared to untreated mice. 120 Hemophilia B is a blood clotting disorder caused by mutations in the clotting factor XI gene. 181 In a Phase I clinical trial, researchers introduced ZFNs to patients intravenously. The editing agents could install the correct clotting factor XI gene into the albumin locus of hepatocytes. The aim of the clinical trial is to produce a permanent secretion of coagulation factor XI in the body ( NCT02695160 ).

Metabolic disorders

Mucopolysaccharidosis is a metabolic disorder caused by the congenital absence of lysosomal enzymes. Furthermore, most mucopolysaccharidoses are autosomal recessive. This disease has seven typical clinical types, and two clinical trials for MPS I and MPS II are currently underway. 182 MPS I is primarily due to a lack of α‐L‐iduronidase, while the shortage of iduronate sulfatase primarily causes MPS II. Angamo Therapeutics Inc. used AAV‐derived vectors to deliver gene‐editing components into hepatocytes. These ZFNs could insert a normal α‐L‐iduronidase gene or iduronate 2‐sulfatase gene into albumin sites to obtain lifetime lysosomal enzyme production capacity ( NCT02702115 , NCT03041324 ). Additionally, several published reports have demonstrated the safety and effectiveness of the CRISPR system in treating MPS I. 183

ADA deficiency is a metabolic disease caused by mutations in the ADA gene. 184 , 185 In a previous clinical study, the patient‐derived HSPC genome was corrected by introducing ADA complementary DNA, and the corrected cells were then autologously transplanted into patients ( NCT01380990 ). It is worth noting that the gene fragment was introduced by lentivirus, which may carry a risk of insertion mutation.

In Type 1 diabetes mellitus, the islet cells that produce insulin in the pancreas are mistakenly attacked by effector T cells, failing to respond to blood glucose changes. Additionally, regulatory T (Treg) cells cannot properly interfere with this false attack, which ultimately leads to the progression of diabetes. Researchers use gene‐editing technology to target the forkhead box P3 ( FOXP3 ) gene in human T cells, inserting a robust enhancer/promoter proximal to the first coding exon. The persistent activation of the FOXP3 gene allows T cells to be artificially intervened to transform into Treg cells. These edited Treg cells have the potential to stop the negative reaction within the pancreas for treating diabetes. The clinical benefits of gene‐editing therapeutics for Type 1 diabetes still require more study. 186

Neurodegenerative disorders

NDs are caused by the loss of neuronal structure or function in the brain and spinal cord, including AD, PD, HD, and amyotrophic lateral sclerosis (ALS). The number of patients gradually increases due to the lack of effective early diagnosis and successful therapeutic intervention. Additionally, as limited by the understanding of the pathogenesis of NDs, the exploitation of therapeutics is extremely challenging. Genetic mutation and the aggregation of misfolded proteins are considered potential pathogenic mechanisms of ND. 187 , 188 Therefore, correcting gene or protein errors based on the gene‐editing platform is promising for exploring and treating NDs. There are thousands of clinical trials on NDs. In addition to a few approved monoclonal antibodies, no ND treatments have displayed promising clinical outcomes. In addition, many results of clinical trials have yet to be verified. 189 Currently, gene‐editing therapeutics for NDs are still in their infancy. Thus, we primarily present some preclinical research progress here.

AD is one of the most common chronic NDs, and its primary pathological features are amyloid plaques and neurofibrillary tangles, which eventually lead to a severe cognitive disorder. 190 Swedish scientists reported that they used the CRISPR‐Cas9 system to correct the mutation of the gene encoding APP in a patient's cells. This change could lead to a treatment for AD. 191 In addition, scientists have generated several antibodies targeting β‐amyloid protein for active or passive immunity. As significant components, apolipoprotein E and PSEN participate in the disease process of AD and can serve as potential targets for the gene‐editing system. 192 HD is a rare ND with an autosomal dominant inheritance caused by an abnormality in the HTT gene. 193 The accumulation of abnormal metabolites (the huntingtin protein) damages the cerebral cortex, leading to mental decline and the loss of athletic ability. 194 Although tetrabenazine is approved for treating HD, the effect of the drug is limited. Current treatment strategies to alleviate HD primarily include directly replacing damaged or lost neurons, knocking out or silencing genes that express abnormal proteins, and reducing the harm caused by the huntingtin protein to protect the surviving neurons. 195 , 196 The main pathological feature of PD involves the loss of dopaminergic neurons in the substantia nigra pars compacta and brain cell death. 197 , 198 As there is no cure for PD, the current conventional treatment strategy is to compensate for the loss of dopamine artificially; however, this approach only relieves the symptoms. A recent study used the CRISPR‐Cas9 system to inactivate the alpha synuclein ( SN CA ) gene encoding alpha‐synuclein, which is related to the formation of Lewy bodies. Compared to unedited cells, edited stem cells can differentiate into dopamine‐producing neurons and do not produce Lewy bodies when subjected to specific chemical stimuli. This work reaffirms the potential of gene editing‐mediated cell replacement therapy in treating NDs. 199 , 200 ALS is characterized by the extensive degeneration of motor neurons in the spinal cord, brain stem, and cerebral cortex. 201 , 202 The current treatment strategy replaces damaged motor neurons with neural stem cells that secrete neurotrophic factors. Mutations in the gene encoding superoxide dismutase 1 are one of the pathological causes of ALS. 203 Researchers from the University of California, Berkeley, verified that the deletion of the superoxide dismutase type 1 ( SOD1 ) gene could improve the retention of motor neurons in a rat model. 204 , 205

To conclude, the lack of feasible treatment options for NDs is temporary. Gene‐editing platforms allow us to understand the occurrence of diseases at the genetic level and to establish more complete disease models.

Ocular disorder

The great potential of using limbal stem cells for treating corneal injury is described above. Gene therapy has also made significant progress in treating hereditary eye diseases. LCA is a major hereditary disease that causes blindness in children, of which LCA10 is the most common and the most serious type. In 2017, the US FDA approved a gene therapy called Luxturna (voretigene neparvovec‐rzyl) to treat patients with LCA2 with RPE65 gene mutations. 206 , 207 , 208 The pathological cause of LCA10 is a mutation of the CEP290 gene (the most common mutation is termed p.Cys998X). 113 In 2020, Allergan and Editas Medicine Inc. initiated a clinical trial to treat LCA10 using AGN‐151587 (EDIT‐101), which could eliminate the mutation in CEP290 using Cas9 effectors, combined with the AAV vector ( NCT03872479 ).

THERAPEUTIC GENOME EDITING: OPPORTUNITIES AND CHALLENGES COEXIST

Genome‐editing technologies increase the potential to treat genetic diseases

According to the latest data from the OMIM Gene Map Statistics, more than 4000 genes will cause morbid phenotypes in humans. 209 A total of millions of people worldwide suffer from genetic disorders. Unfortunately, most of them are unable to receive optimal treatments because of the dearth of therapeutic drugs and diagnostic methods. 210 Traditional drug development to treat rare genetic diseases costs vast amounts of money and time, forcing many pharmaceutical companies to retreat. Exploiting a novel treatment for genetic diseases is urgently needed. 211 , 212 The plight of unmet medical needs is currently expected to be addressed by therapeutic genome editing. Emerging gene‐editing technologies have expanded our abilities to manipulate the gene sequences of eukaryotic cells. 30 , 36 , 58 , 60 , 175 These molecular tools make it possible to cure genetic diseases by correcting or removing the errors within genome sequences.

Notably, therapeutic products based on gene‐editing technologies have offered unique advantages over traditional small molecular drugs and antibodies. 147 A prominent strength of gene therapy is that it can offer a customized therapeutic schedule for individuals who have limited or no treatment options. 212 Of course, personalized clinical procedures may be restricted by regulatory guidelines and have a heavy financial burden. The promising clinical benefits, however, will encourage regulatory authorities to adjust their policies toward these emerging therapies. 213 The treatment strategies for most gene‐editing therapies can be briefly summarized in several steps, including screening, synthesis, delivery, the detection of target gene sequences and industrialized production. 214 Compared to small‐molecule targeted drugs and monoclonal antibodies, nucleic acid sequences are easy to design and less expensive. 215 Additionally, the expanding gene‐editing arsenal has provided many versatile tools to manipulate the gene sequence as needed. 216 Last, the desired gene cargoes and editing tools could be transferred into cells efficiently using various vehicles, including viral or nonviral vectors. 217 Simplified research and development processes can reduce time and vastly accelerate the progress of gene therapy. 218

Several challenges limit the development of clinical applications

Efficiency

In addition to safety, the wide applications of genome editing depend on its efficiency. The common factors affecting editing efficiency include the following: (i) the target cell type and cellular environment, (ii) the optimal choice of gene‐editing tools, (iii) the competition of different cellular DNA repair pathways, and (iv) the technologies for delivering editing components. 219 , 220 , 221 , 222 Of these factors, the efficient delivery of genome editing machinery in vitro and in vivo is the foundation of successful gene‐editing therapeutics. 217 Therefore, we will focus on discussing the development of delivery strategies. Previous studies have developed multiple delivery methods to transfer macromolecules (such as proteins, DNA, siRNA, or mRNA) into the cell. 145 , 223 The current delivery strategies can be classified into two formats: virus‐based delivery systems and nonviral delivery systems (Figure 4 ). 145

A variety of viral vehicles, including AAVs, lentiviruses, adenoviruses, and retroviruses, have been applied to deliver gene‐editing components into target cells of interest. 144 , 224 , 225 , 226 Most currently, AAVs and lentiviruses, depending on some unique advantages, have been widely used in clinical trials. 227 , 228 , 229 Natural AAV vectors have 11 serotypes, which possess inherent tropisms for different tissues. 227 Because of the tropism for the eye, AAV2 has been approved by the FDA for treating degenerative retinal disorders. 230 , 231 Moreover, AAV vectors could reduce the risk of genomic mutation because the gene cargos carried by AAV cannot integrate into the host genome. Although many attractive features of AAVs have been noted, some existing challenges are still awaiting address. The first challenge is the limited packing capacity of AAV vehicles. 232 Most AAV vectors can encode up to 4.4 kb of exogenous DNA, less than other tools. 233 Sometimes, the carrying capacity can accommodate only one CRISPR‐Cas endonuclease gene without other space for sgRNA or donor templates. A feasible solution is using two vectors to deliver the whole system with the potential to reduce efficiency. 234 , 235 The employment of smaller engineered Cas variants is an alternative strategy to tackle the capacity limit and improve efficiency. 236 Second, the preexisting adaptive immunity to natural serotypes hinders the in vivo delivery effectiveness of AAV vectors. 237 However, capsid engineering may circumvent this limitation. 238 , 239 In addition, the transgene delivered by AAVs has long‐term expression in target cells, increasing the editing efficiency but simultaneously posing a potential risk of off‐target toxicity or immunotoxicity. 240

Lentivirus, the replication‐incompetent vector with a 10 kb packing capacity, 241 is capable of introducing therapeutic DNA into primary cells, such as T cells or HSCs. 242 For instance, in four approved CAR‐T therapies, CAR genes were introduced into lymphocytes using lentivirus. 243 , 244 , 245 , 246 However, the random integration of exogenous genes has restricted the usage range of lentivirus. 247 Moreover, some characteristics, including the narrow scope of the target specificity and poor delivery efficiency, are also the current barrier to lentivirus applications. At present, the appearance of novel lentiviral vectors, such as integrase‐defective lentiviral vectors, perhaps provides the potential to address these issues. 248 , 249 , 250 In general, for most virus‐based delivery systems, the existing manufacturing technique cannot adapt to the changing requirements. 251 Technological innovation is critical to improving the efficiency of viral vectors. For example, the potency of AAVs has been enhanced by protein engineering. 252

Nanoparticles have been found to be suitable for delivering genome editors and provide many options for delivery vehicles, including polymer, lipid, and gold nanoparticles. 253 , 254 , 255 Unlike viral vectors that only accommodate cDNA encoding therapeutic effectors, nonviral delivery systems possess a tunable carrying capacity for various transportation objects. 256 For instance, Cas9 RNPs encapsulated in cationic LNPs are engaged by cells via endocytosis and micropinocytosis. 257 A feasible approach led to moderate editing outcomes in a mouse model. 258 Although the editing efficiency of lipid‐mediated nanoparticles is not equivalent to that of virus vectors, nanoparticles present many unique advantages. In addition to their low cost and convenient assembly, one major strength is their transient expression in target cells because nanoparticles cannot induce a long‐term reaction, reducing the risk of off‐target effects or immunotoxicity. However, the injected nanoparticles primarily accumulate in the liver or spleen, causing undesired toxicities, which limits their clinical application. Gold nanoparticles are considered an attractive candidate for tissue‐targeted delivery because of their modifiable surface characteristics. 259 , 260 An alternative strategy to reduce toxicity is electroporation. 261 , 262 This microbiological technology increases the permeability of the cell membrane by using a current pulse, allowing gene‐editing components to enter cells. The straightforward delivery procedure improves the editing efficiency in cells while preserving cell viability as much as possible. 263 However, the scenarios for electroporation use are limited, and in vivo delivery is impractical. Some investigators have tried to combine different delivery methods to improve editing efficiency. 264 Dai et al. established a system to generate CAR‐T cells with immune checkpoint gene knockout using electroporation and AAV6 vectors. These attempts will accelerate the development of delivery systems. 265 In summary, even though current delivery systems have some disadvantages, emerging delivery strategies will offer more options for us to achieve ideal gene editing.

Safety considerations

However, as previously mentioned, an increasing number of clinical applications and tests have demonstrated the availability of gene editing. 153 , 266 , 267 , 268 There are still several technical limitations that hinder the broad clinical utility of genome editing. Scientists have made a great deal of effort to break through these limitations by developing various hybrid strategies.

The safety of therapeutic gene editing is a key concern for investigators and patients. 147 , 269 , 270 The primary security risks arise from the discrepant editing accuracy and immunogenicity of gene‐editing effectors or delivery reagents. 271 Ideal editing outcomes involve the precise installation of desired mutations within the target site without creating any byproducts. However, due to deficient target specificity, DNA cleavage may occur at the wrong genome locations that have similar features to the intended editing sequence. In addition, the DSBs generated by various gene‐editing nucleases generally induce different modes of DNA repair, including NHEJ and/or HDR. 272 In some cases, even if the desired DSBs appear in the target site, the unpredictable and complex repair process may also cause undesired mutagenesis. 221 , 273 For instance, once off‐target events occur in cardiac cells, even the frequency is small, which will cause irreversible problems. 274 To improve the accuracy and precision of gene editing, some researchers have tried to generate new orthologs with greater specificity, 275 such as SpCas9 and xCas9. 25 , 276 , 277 , 278 Another approach is to avoid producing DSBs. 70 , 279 Liu et al. demonstrated that base editors and prime editors could install a single base mutation or small fragment within genome sequences without reliance on DSBs and HDR. 58 , 70 Moreover, it is worthwhile to establish effective methods for monitoring off‐target events in humans. 280 Some current evidence has suggested that in vitro analysis of off‐target editing in primary cells can serve as a guide for in vivo situations. 280

The immunogenic toxicity of editing proteins is frequently mentioned in association with CRISPR‐Cas systems. The engineered Cas nucleases were remolded based on natural Cas proteins derived from some bacteria. 19 If individuals have ever been infected by these pathogens, the engineered editing effectors have the potential to be captured by preexisting antibodies and trigger inflammation and other unknown side effects. 281 , 282 Although some studies have detected specific antibodies and preexisting adaptive immunity to Cas9 in humans, 282 , 283 we still need sufficient evidence to clarify the occurrence mechanism of immunotoxicity caused by preexisting antibodies. 284 Together, these studies are helping to identify more secure proteins for gene editing in the clinic. The immune response to the delivery modality has been discussed in the previous section.

Ethical challenges

Many studies have highlighted the remarkable therapeutic benefits and promising future of gene‐editing therapy. However, we cannot focus only on short‐term successes and ignore the unique ethical challenges. 285 Although germline changes have been completed in plants or some animals, 286 , 287 there is some controversy over human germline alterations. There is no doubt that human germline genome editing must be rigorously regulated. 288 In the absence of sufficient understanding, all scientists should stand in awe of human germline genome editing. Of course, some rational attempts should be allowed under strict supervision.

FUTURE OUTLOOK AND CONCLUSION

A profound revolution and innovation have been driven by gene‐editing technologies in many fields, including agriculture, medicine, biotechnology, and the manufacturing industry. In the last decade, more efficient and versatile gene‐editing platforms have been established and now offer us a powerful tool for investigation and genome engineering in eukaryotic cells. As a research tool, various gene‐editing agents have enabled us to characterize and understand the functions of normal genes. In addition, scientists have the opportunity to screen disease‐causing mutations and elucidate the pathogenesis of some rare genetic diseases. As a diagnostic tool, CRISPR‐Cas systems have been re‐engineered to detect viral nucleic acids, such as SARS‐CoV‐2. The successful application of gene editing in the diagnosis of infectious diseases indicates that CRISPR‐Cas nucleases have the potential to be developed into an accurate and rapid diagnostic tool for other diseases. The most inspiring application of gene editing is in the field of gene or cell therapy. The correction and alteration of disease‐causing gene mutations offer the possibility of treatment or even a permanent cure for some genetic disorders. Emerging gene manipulation tools have addressed many technological issues associated with immunotherapy. For instance, some immunotherapies, particularly CAR‐T therapy, are poised to create a “paradigm shift” in malignant tumours. To date, we have witnessed many promising clinical outcomes and have accumulated increasing clinical experience.

Despite the fact that gene‐editing therapeutics have been subject to tremendous progress in clinical applications, several formidable problems need to be approached before the ultimate aspiration of curing all genetic disorders can be fulfilled. First, scientists continue to increase the accuracy and efficiency of existing gene‐editing agents in parallel with innovations and developments of novel technologies. Additionally, we still emphasize the urgent need for optimal delivery approaches, which are the major barriers to achieving efficient gene manipulation in vivo. Furthermore, bioethicists have stressed that the original intention of genome editing is to correct morbific errors rather than eliminate differences. Therefore, we should be vigilant about the deliberate or unintentional misuse of these customized tools.

Scientific technology offers a profound opportunity to reshape medical treatments. Realizing the full potential of gene‐editing technology depends not only on the efforts of scientists and clinicians but also on the support of the government and other stakeholders. It is foreseeable that gene‐editing technology will provide a novel avenue for health care in the future.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

ETHICS STATEMENT

The authors declare that human ethics approval was not needed for this study.

AUTHOR CONTRIBUTIONS

W.W. conceived and presented the article idea and supervised the whole work. W.Z. collected the data and wrote the first draft of the manuscript. J.Y., Y.Z., and X.H. participated in editing the manuscript. W.W. provided important suggestions for manuscript writing. All authors participated in the work and approved the manuscript for publication.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (81972878 and 82172733), the National Key Research and Development Program of China (2016YFC1303403 and 2020YFC0860200) and the Key Research and Development Program of Sichuan Province (2020YFS0008).

Zhou W, Yang J, Zhang Y, Hu X, Wang W. Current landscape of gene‐editing technology in biomedicine: Applications, advantages, challenges, and perspectives. MedComm. 2022;3:e155. 10.1002/mco2.155

DATA AVAILABILITY STATEMENT

The data included in this study are available upon request from the corresponding author.

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Associated Data

Data Availability Statement

The data included in this study are available upon request from the corresponding author.