CRISPR-Cas12a–assisted PCR tagging of mammalian genes

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.

Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex

“…To ensure that these results were not an artifact of protein overexpression, we analyzed the interaction between endogenous MBRL and RNF185. Using CRISPR-Cas12-mediated genome editing ( Fueller et al., 2020 ), we tagged endogenous MBRL with the mNeonGreen (mNG) fluorescent protein (MBRL-mNG). Western blotting, flow cytometry, and depletion experiments demonstrate that at least one, but not all, MBRL alleles was successfully tagged with mNeonGreen ( Figures 4 C and S4 C).…”

“…MBRL and HRD1 were endogenously tagged with mNeonGreen at their C terminus in HeLa cells using the PCR-tagging technique ( Fueller et al., 2020 ). Cells were selected using Zeocin (100 μg/mL; Invitrogen) for 7 days, after which mNeonGreen-positive cells were single-cell sorted using a BD FACSAria3 or a Beckman Coulter MoFlo Astrios.…”

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

“…A recent study introduced PCR tagging as a simple and time-efficient method for genetic labeling of target proteins ( Füller et al., 2020 ), which makes it a promising labeling tool for quantitative super-resolution microscopy. Using PCR tagging, we generated a stable cell line in which mEos4b was fused C-terminally to the endogenous MET receptor ( Figure 1 A).…”

“…Despite numerous advantages of CRISPR/Cas ( Yamamoto and Gerbi, 2018 ), genetic insertions with standard CRISPR/Cas methods are time intense. Taking advantage of the CRISPR/Cas12a system, a new genome engineering approach was introduced (termed termed polymerase-chain reaction [PCR] tagging) which represents a time-saving method to generate the homologous template required for gene insertion ( Füller et al., 2020 ). The main advantage of this technique is the fast generation of the homology template (PCR cassette) by a single PCR reaction using primers (which provide the homology arms) that can be designed with an online tool ( www.pcr-tagging.com ).…”

“…The DNA cut is then repaired by homology directed repair of the PCR cassette, which labels the protein of interest with the desired tag. This makes PCR tagging a simple and time-efficient alternative for gene labeling with which high labeling efficiencies are achieved ( Füller et al., 2020 ).…”