Open AccessSuppression of HBV replication by the expression of nickase- and nuclease dead-Cas9
Author(s) -
Takeshi Kurihara,
Takasuke Fukuhara,
Chikako Ono,
Seiji Yamamoto,
Kentaro Uemura,
Tomoyoshi Okamoto,
Masaya Sugiyama,
Daisuke Motooka,
Shota Nakamura,
Masahito Ikawa,
Masashi Mizokami,
Yoshihiko Maehara,
Yoshiharu Matsuura
Publication year - 2017
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/s41598-017-05905-w
Subject(s) - cas9 , biology , genome editing , crispr , guide rna , nuclease , cccdna , genome , dna replication , hepatitis b virus , microbiology and biotechnology , virology , genetics , dna , gene , virus , circular dna
Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo . However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro . Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.