PT  - JOURNAL ARTICLE
AU  - Julia Fueller
AU  - Konrad Herbst
AU  - Matthias Meurer
AU  - Krisztina Gubicza
AU  - Bahtiyar Kurtulmus
AU  - Julia D. Knopf
AU  - Daniel Kirrmaier
AU  - Benjamin C. Buchmuller
AU  - Gislene Pereira
AU  - Marius K. Lemberg
AU  - Michael Knop
TI  - CRISPR-Cas12a-assisted PCR tagging of mammalian genes
AID  - 10.1101/473876
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 473876
4099  - http://biorxiv.org/content/early/2020/02/11/473876.short
4100  - http://biorxiv.org/content/early/2020/02/11/473876.full
AB  - Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes