CRISPR-Cas12a-assisted PCR tagging of mammalian genes

J Cell Biol. 2020 Jun 1;219(6):e201910210. doi: 10.1083/jcb.201910210.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Cell Line
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Gene Targeting / methods*
  • Genes, Reporter
  • High-Throughput Nucleotide Sequencing
  • Homologous Recombination
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Oligonucleotides / genetics
  • Polymerase Chain Reaction / methods*
  • RNA, Guide / genetics
  • Transfection

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Luminescent Proteins
  • Oligonucleotides
  • RNA, Guide
  • Cas12a protein
  • Endodeoxyribonucleases