Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre

AbstractGenome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least 6 months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80–90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.

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Short-Term and Highly Efficient Cas9 Expression System Using Adenovirus Vector and Site-Specific Recombinase Cre

10.21203/rs.3.rs-900410/v1 ◽

2021 ◽

Author(s):

Sayaka Nagamoto ◽

Miyuki Agawa ◽

Emi Tsuchitani ◽

Kazunori Akimoto ◽

Saki Kondo ◽

...

Keyword(s):

Hepatitis B ◽

Genome Editing ◽

Expression System ◽

Adenovirus Vector ◽

Virus Genome ◽

Dna Viruses ◽

Site Specific ◽

Tissue Specific ◽

Tissue Specific Promoter ◽

A Cell

Abstract Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least six months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80–90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.

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Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

International Journal of Molecular Sciences ◽

10.3390/ijms221910570 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10570

Author(s):

Yuya Kato ◽

Hirotaka Tabata ◽

Kumiko Sato ◽

Mariko Nakamura ◽

Izumu Saito ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Genome Editing ◽

Adenovirus Vector ◽

Target Sequence ◽

Guide Rna ◽

Guide Rnas ◽

B Virus ◽

X Gene

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

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Aberrant tissue specific expression of the transgene in transgenic mice that carry the hepatitis B virus genome defective in the X gene

Archives of Virology ◽

10.1007/bf01309547 ◽

1993 ◽

Vol 132 (3-4) ◽

pp. 381-397 ◽

Cited By ~ 3

Author(s):

H. Nagashima ◽

M. Imai ◽

Y. Iwakura

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Transgenic Mice ◽

Virus Genome ◽

Specific Expression ◽

Tissue Specific ◽

Tissue Specific Expression ◽

B Virus ◽

X Gene

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Identification of a Hepatitis B Virus Genome in Wild Chimpanzees (Pan troglodytes schweinfurthi) from East Africa Indicates a Wide Geographical Dispersion among Equatorial African Primates

Journal of Virology ◽

10.1128/jvi.76.21.11155-11158.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 11155-11158 ◽

Cited By ~ 21

Author(s):

Jean-Pierre Vartanian ◽

Pascal Pineau ◽

Michel Henry ◽

William D. Hamilton ◽

Martin N. Muller ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

East Africa ◽

Pan Troglodytes ◽

Entire Range ◽

Virus Genome ◽

Eastern Africa ◽

Dna Viruses ◽

B Virus ◽

Geographical Dispersion

ABSTRACT DNAs from four wild chimpanzees (Pan troglodytes schweinfurthi) from eastern Africa were screened for 14 DNA viruses and retroviruses. Between two and three viruses were found in each animal. An entire hepatitis B virus (HBV) genome was amplified and sequenced from samples taken from one animal. This indicates that HBV is distributed across the entire range of chimpanzee habitats.

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Long-term mutation rates in the hepatitis B virus genome

Microbiology ◽

10.1099/0022-1317-81-1-75 ◽

2000 ◽

Vol 81 (1) ◽

pp. 75-83 ◽

Cited By ~ 101

Author(s):

Charles Hannoun ◽

Peter Horal ◽

Magnus Lindh

Keyword(s):

Liver Disease ◽

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Viral Genome ◽

Virus Genome ◽

Serum Samples ◽

Cross Sectional ◽

B Virus

Mutations in the hepatitis B virus (HBV) genome have so far been investigated in cross-sectional or short-term longitudinal studies. Information about long-term changes is lacking due to the difficulty of sampling over long observation periods. In this study, a retrospective approach was used that allowed the analysis of changes in the viral genome from transmission to late stages of infection without the requirement for sampling early during this period. The entire viral genome was sequenced from serum samples of three mothers and their 10 adult children, who presumably had been infected vertically. The emergence of mutations between birth and sampling (mean 26·5 years) was assessed by comparing the individual sequences with the sequence of the strain assumed to have been transmitted. The mean differences from this sequence were 0·02 and 0·28% in seven asymptomatic and one symptomatic hepatitis B e antigen (HBeAg)-positive carriers, respectively, and 0·62 % in five HBeAg-negative carriers. Mutations occurred throughout the genome and 88% of the mutations caused amino acid substitutions spread over all genes. In HBeAg-negative carriers, the number of nucleotide and amino acid changes was independent of the severity of liver disease and, except the 1762AGG1764→TGA changes, no specific mutation was associated with liver disease. In conclusion, by using a novel method it was found that the entire HBV genome is extremely stable over long periods of time during the HBeAg-positive phase if the immune response (inflammation) is weak, whereas an average of 20 mutations emerged after development of hepatitis and/or loss of HBeAg without association with clinical outcome.

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