Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system.

ABSTRACT: The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle,

SUBMITTER: Li Y 

PROVIDER: S-EPMC6269370 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature