Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.

ABSTRACT: CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a sel

SUBMITTER: Manna PT 

PROVIDER: S-EPMC6899835 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature