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


CONCLUSIONS
Our studies offered simple and efficient methods to manipulate PRV.
State Key Laboratory of Agrobiotechnology and College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
Aotian
A
Xu
Chao
C
Qin
Yue
Y
Lang
Mingyue
M
Wang
Mengyang
M
Lin
Chuang
C
Li
Rui
R
Zhang
Jun
J
Tang
D064113:CRISPR-Cas Systems; D004337:Drug Carriers; D055879:Gene Knock-In Techniques / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D016679:Genome, Viral; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D013997:Time Factors
D004337:Drug Carriers