| Autor: |
Gislene Pereira, Bahtiyar Kurtulmus, Krisztina Gubicza, Matthias Meurer, Konrad Herbst, Benjamin C. Buchmuller, Julia Fueller, Julia D. Knopf, Michael Knop, Daniel Kirrmaier, Marius K. Lemberg |
| Jazyk: |
angličtina |
| Rok vydání: |
2018 |
| Předmět: |
|
| DOI: |
10.1101/473876 |
| Popis: |
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 |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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