cccDNA is the major determinant of viral persistence.
CRISPR/Cas nucleases have been recently shown to introduce double-stranded DNA breaks into HBV cccDNA.
The inflicted damae results predominantly in erroneous repair of cccDNA by NHEJ.
Conclusion:CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonsrtate that inhibiting NHEJ impairs cccDNA degradation.
Hepatitis B virus(HBV) belongs to the family Hepadnaviridae. The virial genome of the enveloped HBV particle comprises a circualr, partially double-stranded DNA that is synthesized by reverse transcriptase.
Upon entry, HBV virons are coated, the relaxed circular DNAis transported into the nucleus where it is repaired to form cccDNA. cccDNA is the key component of that HBV life cycle that serves as a template for transcription of all viral mRNAs including pre-genomic RNA (pgRNA)
Current approved therapies suppress HBV replication, but do not target cccDNA, which persists in nuclei of ingected hepatocytes.
Now use CRISPR/Cas9 to target HBV cccDNA in infected cells.
Mammalian cells repair damaged DNA:
- Error-prone canonical non-homologous end-joining (NHEJ) (predominant form of DSB repair) operatng in all phases
- Canonical: Induction of DNA DSBs leads to the recruitment of KU70/80 heterodimer to the ends of damaged DNA; followed by interaction with catalytic subunit of DNA-dependent protein kinase(DNA-PKcs) -- at the two broken DN ends to generate the DNA-PK holoenzyme. DNA-PK enzyme acts as a scaffold protein that tethers the broken ends together. Finally, the protein phophotylation reactions ultimately results in the excision of single-stranded overhangs of broken ends and ligation of DNAA ends by the complex of ligase.
- Error-free homologous recombination (HR) only operating in the late S and G2 phases.
- MRE11-RAD50-NGS1 complex first recognizes the DSBs and trims DNA ends to form 3′-overhangs of single-stranded DNA (ssDNA). RPA stabilizes the ssDNA ends to prevent formation of secondary structures. Then substituted by RAD51, a key component of HR that coats ssDNA to form nucleoprotein filaments essential for the homology search and strand exchange.
CRISPR/Cas9 is not efficient enough
With over 30% of total cccDNA remaining in the cells 3 weeks after CRISPR/Cas9 transduction, and 10% 5 weeks after.
The on-target mutations corresponding to the sgRNAs sequence will mostly escape CRISPR/Cas9 activity and will not be cleaved repetitively due to limited mismatch tolerance of CRISPR/Cas9.
Single mismatches in target region, nucleotide indels or dewer than 6 mismatches in distal regions of sgRNA are well tolerated by most typeII CRISPR/Cas9 system.
In life cell, the majority of HBV cccDNA genomes remianed intact. The reason could be:
- A portion of cccDNA is not accessible to Cas9 proteins.
- cccDNA is cleaved by CRISPR/Cas9 but is repaired by HR without on-target mutagenesis.
So it seems DNA repair is in contract to DNA cleavage
We analyzed anti-HBV activity and cccDNA cleavage outcomes of CRISPR/Cas9 targeting and enhancing NHEJ (with 3-aza) and HR (with L755) or suppressing NHEJ and HR using NU7026, Ad4E1B, or B02.
Toxicity of small molecules and effects on viability and cell cycle
3aza decreased viability of cells.
HBV was previously shown to dysregulate the G1 to S transition. Incubation with NU7026 and B02 mild increase the proportion of cells in G1/G0, while treatment with either NU7026 or B02 decreased the proportions of cells in S-phase and G2/M compared to controls.
Analysis of apoptisus did not reveal significant differences between experimental grousp and control.
Genotoxicity of NU7026 and B02 was assessed by immunostaining and counting foci of yH2AX, Ser-139 phosphorylated form of H2AX protein, a reliable marker of DNA damage response in the cells.
- Few yH2AX foci (0.09 ± 0.11) were detected in control cells.
- incubation with B02 resulted in generation of small yH2AX foci (2.78 ± 0.98 per cell).
- NU7026 did not induce yH2AX foci formation
Design of CRISPR/Cas9
Use 3 sgRNSs targeting core/pre-core, EnhI, and X-gene regions.
Anti-HBV activity of CRISPR/Cas9 in an altered NHEJ/HR environment
Thus, CRISPR/Cas9 systems were very effective in cleaving HBV cccDNA and integrated HBV DNA, as indi- cated by all parameters tested.
Among all NHEJ/HR inhibitors and enhancers tested, only NU7026 significantly affected CRISPR/Cas9-mediated anti-HBV activity.
Inhibition of NHEJ by NU7026 results in CRISPR/Cas9-mediated hyper-editing of HBV cccDNA
Compared to DMSO control groups, NU7026 enhanced deletion formation upon CRISPR/Cas9 transfection, with deletion frequency as high as 180–200 per 1,000 reads