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 and cloning-free strategy for genomic editing of mammalian cells. The desired integration fragment is produced as a PCR product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9/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 selection of key membrane trafficking proteins. The lack of any donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.