CRISPR-Cas12a–assisted PCR tagging of mammalian genes
| Autor: | Konrad Herbst, Julia D. Knopf, Julia Fueller, Marius K. Lemberg, Bahtiyar Kurtulmus, Gislene Pereira, Benjamin C. Buchmuller, Daniel Kirrmaier, Michael Knop, Krisztina Gubicza, Matthias Meurer |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
CRISPR-Associated Proteins
Oligonucleotides Locus (genetics) Computational biology Biology Transfection Polymerase Chain Reaction Homology (biology) Cell Line Tools 03 medical and health sciences 0302 clinical medicine Plasmid Bacterial Proteins Genes Reporter Genetics CRISPR Humans Clustered Regularly Interspaced Short Palindromic Repeats Homologous Recombination Gene Alleles Cells Cultured 030304 developmental biology Cancer Organelles 0303 health sciences Endodeoxyribonucleases Oligonucleotide High-Throughput Nucleotide Sequencing Cell Biology Luminescent Proteins Terminator (genetics) Gene Targeting CRISPR-Cas Systems Homologous recombination 030217 neurology & neurosurgery RNA Guide Kinetoplastida |
| Zdroj: | The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| Popis: | Fueller et al. describe a simple one-step procedure for quick and scalable chromosomal tagging of genes in mammalian cells using PCR products that contain all elements necessary for Cas12a tagging: homology arms and a guide RNA gene for chromosomal insertion, the tag (such as GFP), and a selection marker. 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 artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein–tagged genes. |
| Databáze: | OpenAIRE |
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