Frontiers in genome editing · 2026
Combined RNA- and DNA-targeting CRISPR ribonucleoproteins enhance suppression of HBV transcripts.
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Banik Puja, Hill Addison C, Schank Madison B, Zhang Yi, Zhao Juan, Pyburn Jaeden S, Orfield Holly, Wu Yuezu, Hajipour Mohammad J, El Gazzar Mohamed, Ning Shunbin, Moorman Jonathan P, Yao Zhi Q, Wang Ling
Аннотация
INTRODUCTION: Chronic hepatitis B virus (HBV) infection remains a major global health problem and there is no curative treatment currently available. RNA-targeting CRISPR systems offer a potential alternative to DNA-editing nucleases by enabling direct degradation of viral transcripts without introducing genomic breaks. In this report, we evaluated the antiviral activity of synthetic guide RNA (gRNA)/Cas13d ribonucleoproteins (RNPs) targeting HBV RNA and a combination use with DNA-targeting RNPs.
METHODS: We designed 10 gRNAs targeting conserved regions across the HBV transcriptome followed by screening for best candidate gRNA for Cas13d. To enhance antiviral efficacy, we evaluated multiplex RNA targeting using combined gRNA6+gRNA7/Cas13d RNPs, and combined RNA-targeting gRNA7/Cas13d with previously validated HBV DNA-targeting gRNA9/Cas9 and gRNA4/Cas12a RNPs for HBV treatment.
RESULTS: Amongst these 10 gRNAs, gRNA7/Cas13d exhibited selective knockdown of its specific target RNA transcript without inducing a broad suppression of total HBV DNA, mRNAs, and proteins. The antiviral effect of gRNA7/Cas13d RNP was transient, diminishing by Day 3 post the treatment. The combined gRNA6+gRNA7/Cas13d treatment also did not further induce global HBV suppression compared with single gRNAs, although gRNA7/Cas13d still showed a robust target-specific RNA cleavage during the combined treatment in HBV cellular models. While gRNA9/Cas9 and gRNA4/Cas12a exhibited the dominant antiviral effects, either alone or in combination, leading to substantial reductions in HBV replication and expression, the addition of gRNA7/Cas13d further enhanced the suppression of the gRNA7-specific HBV RNA transcript, achieving up to an 80% reduction in the target RNA level.
DISCUSSION: Collectively, these findings demonstrate that gRNA7/Cas13d can mediate target-specific HBV RNA cleavage but has limited overall antiviral activity when used alone as a single-agent; however, the combination of DNA- and RNA-targeting CRISPR-RNPs may provide a complementary approach for enhancing target-specific HBV RNA suppression.
METHODS: We designed 10 gRNAs targeting conserved regions across the HBV transcriptome followed by screening for best candidate gRNA for Cas13d. To enhance antiviral efficacy, we evaluated multiplex RNA targeting using combined gRNA6+gRNA7/Cas13d RNPs, and combined RNA-targeting gRNA7/Cas13d with previously validated HBV DNA-targeting gRNA9/Cas9 and gRNA4/Cas12a RNPs for HBV treatment.
RESULTS: Amongst these 10 gRNAs, gRNA7/Cas13d exhibited selective knockdown of its specific target RNA transcript without inducing a broad suppression of total HBV DNA, mRNAs, and proteins. The antiviral effect of gRNA7/Cas13d RNP was transient, diminishing by Day 3 post the treatment. The combined gRNA6+gRNA7/Cas13d treatment also did not further induce global HBV suppression compared with single gRNAs, although gRNA7/Cas13d still showed a robust target-specific RNA cleavage during the combined treatment in HBV cellular models. While gRNA9/Cas9 and gRNA4/Cas12a exhibited the dominant antiviral effects, either alone or in combination, leading to substantial reductions in HBV replication and expression, the addition of gRNA7/Cas13d further enhanced the suppression of the gRNA7-specific HBV RNA transcript, achieving up to an 80% reduction in the target RNA level.
DISCUSSION: Collectively, these findings demonstrate that gRNA7/Cas13d can mediate target-specific HBV RNA cleavage but has limited overall antiviral activity when used alone as a single-agent; however, the combination of DNA- and RNA-targeting CRISPR-RNPs may provide a complementary approach for enhancing target-specific HBV RNA suppression.