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Biotechnol Lett. 2015 Jun;37(6):1265-72. doi: 10.1007/s10529-015-1796-2. Epub 2015 Feb 28.

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

Author information

1
State Key Laboratory of Agrobiotechnology and College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.

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.

PMID:
25724716
DOI:
10.1007/s10529-015-1796-2
[Indexed for MEDLINE]

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