Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote.

ABSTRACT: Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to genera

SUBMITTER: Raveux A 

PROVIDER: S-EPMC5314402 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature