In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining.

ABSTRACT: The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 ce

SUBMITTER: Geisinger JM 

PROVIDER: S-EPMC4856974 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature