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A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system

Author:
Xu, Aotian, Qin, Chao, Lang, Yue, Wang, Mingyue, Lin, Mengyang, Li, Chuang, Zhang, Rui, Tang, Jun
Source:
Biotechnology letters 2015 v.37 no.6 pp. 1265-1272
ISSN:
0141-5492
Subject:
DNA repair, Suid herpesvirus 1, gene therapy, genes, host range, plasmids, vaccine development
Abstract:
OBJECTIVES: The broad host range of pseudorabies virus (PRV) and large capacity for foreign DNA make it a promising vector for the development of vaccines and agents of gene therapy. RESULTS: We show that up to 100 % viral gene disrupting efficiency was achieved by simple co-transfection of the purified PRV genomes with the clustered regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) into cells. Furthermore, CRISPR/Cas9-mediated knock-in of >4-kb-long DNA cassettes into the PRV genome at a positive rate of 50 % by a homology-independent DNA repair mechanism without constructing homology arms. This approach requires only a simple plasmid construction and is applicable to knock-in of other foreign genes. CONCLUSION: Our studies offered simple and efficient methods to manipulate PRV.
Agid:
1331181