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 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.
Highlights
Whilst offering advantages over ectopic expression, endogenous protein tagging is costly in terms of time and resources which can limit its application
Knockouts can be created rapidly and by relying on error-prone non-homologous end joining (NHEJ) to generate insertions or deletions. Short sequences such as small epitope tags can be introduced by homologydirected repair (HDR), using readily synthesised single-stranded oligodeoxynucleotide donors with short homology arms of 50-70 bp flanking the double strand break (DSB) site.[4]
We have achieved comparable results in HeLa, HEK293-T and NRK cells suggesting that our approach will be broadly applicable to mammalian model cell systems
Summary
Whilst offering advantages over ectopic expression, endogenous protein tagging is costly in terms of time and resources which can limit its application. The CRISPR/Cas[9] system has radically simplified genome editing in mammalian cells.[1,2,3] Knockouts can be created rapidly and by relying on error-prone non-homologous end joining (NHEJ) to generate insertions or deletions (indels) Short sequences such as small epitope tags can be introduced by homologydirected repair (HDR), using readily synthesised single-stranded oligodeoxynucleotide donors with short homology arms of 50-70 bp flanking the double strand break (DSB) site.[4] fast, this approach does not allow incorporation of longer sequences such as fluorescent protein tags and requires the screening of a number of single cell derived colonies. Four example fusion-proteins are presented, each requiring a different site of tag integration and each showing a distinct but well characterised localisation within the endomembrane system
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
