Pseudorabies virus can escape from CRISPR-Cas9-mediated inhibition

Peng, Zhiyuan; Ouyang, Ting; Pang, Daxin; Ma, Teng; Chen, Xinrong; Guo, Ning; Chen, Fuwang; Yuan, Lin; Ouyang, Hongsheng; Ren, Linzhu

doi:10.1016/j.virusres.2016.08.001

Cited by 33 publications

(33 citation statements)

References 30 publications

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“…A large fragment may not insert as easily as a short one, which may result in a low efficacy of homology-independent DNA repair. For virus escape from CRISPR, our recent study as well as one conducted by another group have demonstrated that viruses can escape CRISPR easily (35,36), and we further demonstrated that multiple sgRNAs completely abolished the production of infectious viruses from cells (35).…”

Section: Discussionsupporting

confidence: 64%

CRISPR/Cas9‐mediated 2‐sgRNA cleavage facilitates Pseudorabies virus editing

et al. 2018

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Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

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Section: Discussionsupporting

confidence: 64%

CRISPR/Cas9‐mediated 2‐sgRNA cleavage facilitates Pseudorabies virus editing

et al. 2018

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“…CRISPR/Cas9 inactivates the virus by introducing DSBs in its DNA that after repair by NHEJ causes mutation in the virus DNA, some subtypes escape, remain viable and are not recognize by the gRNA. Escape mutants evolve by insertion, deletion, or substitution at the target sites of Cas9 . Targeting viral genome at multiple sites can limit the formation of escape mutants.…”

Section: Limitations Of Crispr/cas9 Tool In Combating Oncovirusesmentioning

confidence: 99%

“…Escape mutants evolve by insertion, deletion, or substitution at the target sites of Cas9. 110 Targeting viral genome at multiple sites can limit the formation of escape mutants.…”

Section: Limitations Of Crispr/cas9 Tool In Combating Oncovirusesmentioning

confidence: 99%

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses

Gilani

Shaukat

Rasheed

et al. 2018

Journal of Medical Virology

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It is evidenced that 20% of all tumors in humans are caused by oncoviruses, including human papilloma viruses, Epstein-Barr virus, Kaposi sarcoma virus, human polyomaviruses, human T-lymphotrophic virus-1, and hepatitis B and C viruses. Human immunodeficiency virus is also involved in carcinogenesis, although not directly, but by facilitating the infection of many oncoviruses through compromising the immune system. Being intracellular parasites with the property of establishing latency and integrating into the host genome, these viruses are a therapeutic challenge for biomedical researchers. Therefore, strategies able to target nucleotide sequences within episomal or integrated viral genomes are of prime importance in antiviral or anticancerous armamentarium. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a powerful genome editing tool. Standing out as a precise and efficient oncoviruses method, it has been extensively applied in recent experimental ventures in the field of molecular medicine, particularly in combating infections including tumor inducing viruses. This review is aimed at collating the experimental and clinical advances in CRISPR/Cas9 technology in terms of its applications against oncoviruses. Primarily, it will focus on the application of CRISPR/Cas9 in combating tumor viruses, types of mechanisms targeted, and the significant outcomes till date. The technical pitfalls of the CRISPR/Cas9 and the comparative approaches in evaluating this technique with respect to other available alternatives are also described briefly. Furthermore, the review also discussed the clinical aspects and the ethical, legal, and social issues associated with the use of CRISPR/Cas9.

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“…Interestingly, while most InDels contributing to escape at non-coding regions were a single base pair, three base pair InDels were observed when the target was within an HIV-1 coding region; i.e., the InDel event may preserve the HIV-1 open reading frame but destroy the CRISPR gRNA sequence homology [42]. The occurrence of InDel escape mutations is a consequence of NHEJ DNA repair and so may also occur for any DNA virus or retrovirus and, indeed, was recently reported for the pseudorabies herpesvirus [46]. NHEJ is almost always the dominant mode of DNA repair [47, 48] and is the desired pathway of repair of CRISPR/Cas9-generated DSBs, because the purpose of CRISPR/Cas9 is to introduce mutations and inactivate viruses.…”

Section: The Problem Of Viral Escapementioning

confidence: 99%