Efficient silencing of hepatitis B

virus S gene through CRISPR-

mediated base editing.
Contributors : Hao Zhou, Xiaomei Wang, Clifford J. Steer, Guisheng Song, Junqi Niu
HEPATOLOGY COMMUNICATIONS
Published by Wiley Online Library

Submitted by : Divya
Bsc. Life sciences
Roll no. – 2020/468
 Hepatitis B Virus and CRISPR
• Hepatitis B virus (HBV) infection is a major global cause
of liver disease and hepatocellular cancer.

• Some drugs have been approved for HBV infection,

but only a few patients could achieve a functional cure.

• CRISPR/Cas9 : It has been used to precisely edit HBV genome through double stranded
break (DSB) but that causes chromosomal instability in host cells.

• CRISPR Cytidine base editors (CBE) : Silence genes by generating a premature stop
codon with no off target effect.
(Image taken from: narayanahealth.org )
Steps in CRISPR Cytidine Base Editing
Vector Sequence Direct
Cell culture,
construction analysis of S sequencing for
lentiviral vector
and HBV gene of on-target and
production and
specific sgRNA different HBV off target
transduction.
design. genotypes editing

Detection of
Reverse HBsAg by ELISA
Immuno- Northern Blot
transcription (Enzyme linked
fluorescence Analysis
PCR assay immuno-
sorbent assay.
 Experimental Methodology
• A single guide RNA named gRNA_S was designed to edit 30th codon of HBV S gene by selecting
conserved sequence of S gene coding strand.
• LentiAncBE4max-BLAST expression vector was constructed by insertion of AncBE4max
sequence into plasmid LentCas9- BLAST.
• Human embryonic kidney HEK293T and hepatoma PLC/PRF/5 cell lines were used to express
CBE.
• HEK293T cells were plated and transfected with vector, Cas9 and gRNA_S. After 24 hours cell
supernatant was collected, filtered and stored.
• PLC/PRF/5 cells were transduced with lentiviral vector and LV-Cas9. Then these cells were
transduced with sgRNA and harvested.
• 11 possible off target sites in cell lines and sgRNA ->Identified->Amplified-> PCR and Sanger
sequencing.
• For HBsAg detection – RNA was extracted from cells and subjected to : Northern blotting,
ELISA, immunofluorescence and RT-PCR.
 RESULTS

1) Design of sgRNA capable of introducing 2) Silencing of HBV S gene by CBE mediated base
premature stop codon in HBV S gene editing (CAG to TAG editing in cells).

3) 71% cells developed a premature stop codon 4) 92% reduction in HBsAg secretion in cells revealed
within S gene. by ELISA.
5) Levels of HBs messenger RNA are significantly 6) Intracellular HBsAg was also reduced by 84%,
decreased, revealed by Northern blot. revealed by immunostaining of HBsAg.

7) Sequencing revealed that no editing of C or G base on predicted off target sites

of host genome was detected, hence no off target effects.

8) PCR and Sequencing confirmed that 95%, 93%, 93%, 9% and 72% S gene sequences of
HBV genotypes B, C, F, and H had binding site of sgRNA.
 Discussion
• Loss of HBsAg protein is the only functional cure of HBV infection.
• CRISPR base editors could mediate C to T base substitution in genome by
introducing premature stop codons in HBV S gene.
• This technique has no off target effect hence maintains chromosomal
stability.

• Limitation : On target editing is only by Sanger sequencing. Next

generation sequencing can provide more accurate data.
• Further studies are needed for in vivo gene editing.

Conclusion : CRISPR mediated base editing is an efficient approach to

silence HBV S gene .
It can have the future potential in curing chronic HBV infection.
 Bibliography
• Zhou H, Wang X, Steer CJ, Song G, Niu J. Efficient silencing of hepatitis B virus S gene through CRISPR-
mediated base editing. Hepatol Commun. 2022;6:1652–1663.
https://doi.org/10.1002/hep4.1933
• Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-
stranded DNA cleavage. Nature 533, 420–424 (2016).
https://doi.org/10.1038/nature17946
• Kuscu C, Parlak M, Tufan T, Yang J, Szlachta K, Wei X, Mammadov R, Adli M. CRISPR-STOP: gene silencing
through base-editing-induced nonsense mutations. Nat Methods. 2017 Jul;14(7):710-712. doi:
10.1038/nmeth.4327. Epub 2017 Jun 5. PMID: 28581493.
https://doi.org/10.1038/nmeth.4327
• https://youtu.be/4YKFw2KZA5o
• https://youtu.be/IiPL5HgPehs
• https://www.narayanahealth.org/blog/world-hepatitis-day/amp/
• https://scholar.google.com
• https://www.ncbi.nlm.nih.gov
• https://youtu.be/0W-_BmrdH-M