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FEBS J. 2014 Apr;281(7):1717-25. doi: 10.1111/febs.12735. Epub 2014 Feb 26.

Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting.

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MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing, China.


The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a versatile RNA-guided mammalian genome modification system. One-step generation of mouse genome targeting has been achieved by co-microinjection of one-cell stage embryos with Cas9 mRNA and small/single guide (sg)RNA. Many studies have focused on enhancing the efficiency of this system. In the present study, we report that simultaneous use of dual sgRNAs to target an individual gene significantly improved the Cas9-mediated genome targeting with a bi-allelic modification efficiency of up to 78%. We further observed that the target gene modifications were characterized by efficient germline transmission and site-dependent off-target effects, and also that the apolipoprotein E gene knockout-mediated defects in blood biochemical parameters were recapitulated by CRISPR/Cas9-mediated heritable gene modification. Our results provide a dual sgRNAs strategy to facilitate CRISPR/Cas9-mediated mouse genome targeting.


Cas9; RGEN; dual sgRNAs; gene targeting; mouse

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