TABLE 1.

Summary of studies utilizing the CRISPR/Cas9 system for HBV disruption and inactivation.

Expression vector	Target regions	HBV models	Evaluation methods	Efficiency	Off target and toxicity	References
Lentivirus	ENII-CP/X, Pre-C, X, X/P	HepG2-NTCP/Cas9, HepAD38, HepG2.2.15	HBcAg immunofluorescence (IF), cccDNA PCR-based sequencing, Sanger sequencing versus NGS	8 to 10-fold in HBcAg reduction, >90% HBV DNA was cleaved, indel mutations in PCR products	IFN-induced +, Cas9-induced cytotoxicity	Seeger and Sohn (2014), Seeger and Sohn (2016)
Plasmids	Pre-S1/2, S, P, P, XCp, PCE	Huh-7/HBV plasmid, HDI-HBV model	HBsAg ELISA, HBsAg/HBcAg IF, Plasmid express few cccDNA, 25.6%, 27.8% indels by T7E1	77%–96% HBsAg suppression, 25.6%, 27.8% indels by T7E1	No cytotoxicity, in vitro and in vivo	Lin et al. (2014)
Plasmids, pX330	X/P, X, C, C/P	Huh-7, HepG2.2.15, cccDNA mouse model	HBsAg/HBeAg ELISA and WB, cccDNA by Southern blot and PCR	60% HBsAg suppression rate, 60%–75% cccDNA reduction	Not evaluated, in vitro and in vivo	Dong et al. (2015)
Plasmids, pX330	Pre-S1/2, S/P, X/P, P, C, C/P	HepG2/HBV plasmid, HDI-HBV model	HBsAg/HBeAg ELISA, HBV DNA Southern blot and qPCR, 11% mutagenesis by T7E1	11% mutagenesis by T7E1, 100-fold HBV DNA inhibition	Cell viability by CCK8 kit	Liu et al. (2015)
Plasmids, Lentivirus	S, C, X, P	HepG2, Hep-NTCP, HDI-HBV NRG mice	HBV s, e, c Ags by ELISA and IF, pgRNA by PCR, cccDNA by SB, 77%–95% decrease in HBV DNA	>60% HBsAg suppression rate, 77%–95% decrease in HBV DNA	Surveyor assay and deep sequencing	Ramanan et al. (2015)
Plasmids	S, S/P	HBV cell culture, HBV mouse model	HBsAg ELISA and IF, HBV DNA by qPCR, HBV DNA suppression/mutations	HBsAg reduced in vitro and in vivo, HBV DNA suppression/mutations	Cell viability, not evaluated	Zhen et al. (2015)
Lentivirus	S, C, RT/YMDD	HepAD38, HepaRG	HBsAg ELISA, HBV DNA by real time qPCR, 90% cccDNA reduction, 99% HBV DNA inhibition	90% cccDNA reduction, 99% HBV DNA inhibition	No cytotoxicity, by Promega kit	Kennedy et al. (2015)
Plasmids	Pre-S2, S, P, Pre-C, C, X	Huh7/HBV1.3 plasmid, HepAD38	HBsAg/HBeAg ELISA, PCR-DNA sequencing	>80% HBsAg/HBeAg reduction, HBV (ccc)DNA was cleaved	No cytotoxicity, by MTT	Wang et al. (2015)
Lentivirus	S/P, X/P	HBV reporter plasmid, HepG2.2.15, Hep-NTCP	RFP/GFP fluorescence, HBsAg ELISA/T7E1 assay	Successfully target HBV-sequences, 40%–90% indels/HBV inactivation	Cytotoxicity, not assessed	Karimova et al. (2015)
All-in-one vector/Cas9n	S, X, C	HepG2/HBV plasmid	HBsAg/HBeAg ELISA, Miseq deep sequencing	HBV antigens were suppressed, HBV DNA was cleaved	No off-target mutations	Sakuma et al. (2016)
Plasmids, pX330	S, X	HepG2Huh7/HBV1.3, M-Tg HBV mice	HBsAg ELISA, HBcAg IF/WB, HBV DNA by SB and qPCR	50% HBsAg/HBeAg reduction, HBV DNA suppressed	Cytotoxicity, not assessed	Zhu et al. (2016)
Plasmid pX459, integrated HBV, AAV	S, S/P, X/P, C/P	HepG2.A64 cell line, HDI-/Tg-HBV mice	HBsAg/HBeAg ELISA, HBsAg IF, HBV DNA by qPCR and sequencing	99.9% HBsAg reduction, HBV (ccc)DNA suppressed	Cell viability OK, by CCK-8	Li et al. (2016), Li et al. (2017a), Li et al. (2018a)
ssAAVs	S/P	HepG2-NTCP, SaCas9, HepG2.2.15	HBsAg ELISA, HBV mRNA ddPCR, T7E1 assay, NGS	50%–95%% HBsAg reduction, 46%–61% indels, cccDNA inhibition	No unintended sequence change	Scott et al. (2017)
Cas9 mRNA/gRNA-LLNs	S/P, X/P, pre-C	HepGAD38, HDI-HBV mice	HBsAg/HBeAg ELISA, T7E1 assay, DNA sequencing	Significant HBsAg/HBeAg reduction, HBV mRNA and cccDNA inhibition	No indel, off-target effect	Jiang et al. (2017)
AAV8, SaCas9	S/P, X/P, Pre-C, C/P	HepG2.2.15, HepAD38, HDI-HBV mice	HBsAg/HBeAg ELISA, Deep DNA sequencing	>80% HBsAg/HBeAg reduction, HBV pgRNA and cccDNA inhibition	Undetectable mutations	Liu et al. (2018)
HCAdV, multiplex	P (RT), XCp, RNase H	HepG2.2.15, HepG2-NTCP	HBsAg/HBeAg ELISA, T7E1, RNA/DNA qPCR	54%–76% HBsAg/HBeAg reduction, 39%–78% HBV DNA inhibition	No mutations	Schiwon et al. (2018)
Lentiviral, SpCas9/StCas9	pre-C/C, Enh1, X	HepG2-1.1merHBV, HepG2-1.5merHBV	HBV DNA and cccDNA, HBV pgRNA qPCR	90% HBV cccDNA reduction, Significant HBV pgRNA inhibition, Suppressed HBsAg, HBcAg	Cell viability OK	Kostyushev et al. (2019a), Kostyushev et al. (2019b), Kostyusheva et al. (2019)
BE lentiviruses	S, P, C, X	HBV-HEK293T cells, HepG2.2.15, HepG2-NTCP	Sanger and MiSeq sequencing, HBsAg ELISA, qPCR	>50% base-editing (BE) efficacy,60% HBV DNA inhibition	No genome mutation	Yang et al. (2020)
AAV2 vector	S, P, C, X	HepG2.2.15, HepG2-NTCP, humanized chimeric mice	HBsAg ELISA, qPCR	Suppressed HBsAg, HBcAg, HBV DNA/cccDNA in vivo	No cytotoxicity	Kayesh et al. (2020)
lentiviral vector	S, C, X, P	HepG2-NTCP-C4-iCas9, PHHs	whole genome sequencing, HBV DNA/RNA qPCR, NGS	2-log HBV cccDNA reduction, 50% cccDNA inhibition in PHHs	Cell viability, Not assessed	Murai et al. (2022)
gRNA/Cas9, RNPs	S, C, X, P	HepG2-NTCP, PHHs	HBsAg/HBeAG ELISA, qRT-PCR, SB, DNA/RNA sequencing	Indel formation, generate episomal variants	Cell viability, Not assessed	Martinez et al. (2022)
AAV2 and LNP-based Cas9, Cas12 RNPs Synthetic gRNA, synthetic Cas9 RNPs	Pre-C S, C, X, P	HepG2-NTCP-30 HepG2.2.15, HepDE19, HepG2-NTCP	HBV DNA and cccDNA HBsAG/HBeAG ELISA, HBV (ccc)DNA qPCR	60%–80% inhibition of HBV DNA and cccDNA LNP-RNPs are more efficient than AAV2 approach 50% HBsAg reduction, 95% cccDNA inhibition	No cytotoxicity No cytotoxicity	Suzuki et al. (2021) Zhang et al. (2023)