Tissue- and time-directed electroporation of CAS9 protein-gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration.

ABSTRACT: A rapid method for temporally and spatially controlled CRISPR-mediated gene knockout in vertebrates will be an important tool to screen for genes involved in complex biological phenomena like regeneration. Here we show that in vivo injection of CAS9 protein-guide RNA (gRNA) complexes into the spinal cord lumen of the axolotl and subsequent electroporation leads to comprehensive knockout of Sox2 gene expression in SOX2+ neural stem cells with corresponding functional phenotypes from the gene knockout. This is particularly surprising considering the known prevalence of RNase activity in cerebral spinal fluid, which apparently the CAS9 protein protects against. The penetrance/efficiency of gene knockout in the protein-based system is far higher than corresponding electroporation of plasmid-ba

SUBMITTER: Fei JF 

PROVIDER: S-EPMC5744710 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature