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

Published version
Peer-reviewed

Type

Article

Change log

Authors

Davis, luther 
Robinson, margaret 

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 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 selection of well characterised membrane trafficking proteins. The lack of 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.

Description

Keywords

CHoP-In, CRISPR, endogenous tagging, genome editing, mammalian cells, CRISPR-Associated Protein 9, CRISPR-Cas Systems, DNA Breaks, Double-Stranded, Gene Editing, HeLa Cells, Humans, RNA, Guide, CRISPR-Cas Systems

Journal Title

Traffic

Conference Name

Journal ISSN

1398-9219
1600-0854

Volume Title

Publisher

Wiley-Blackwell
Sponsorship
Wellcome Trust (086598/Z/08/Z)
Wellcome Trust (100140/Z/12/Z)
The CIMR is supported by Wellcome Trust Strategic Award 100140. LJD is supported by a BBSRC industrial CASE studentship with GSK Research and Development Ltd. MSR and PTM are supported by a Wellcome Trust PRFassociated Programme Grant to MSR (086598)