CRISPR-Cas12a-assisted PCR tagging of mammalian genes

Abstract

<jats:title>Abstract</jats:title><jats:p>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</jats:p>

Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation

“…A critical step in the analysis of membrane protein complexes is to combine efficient one-step affinity purification of proteins expressed at physiological levels. We therefore endogenously tagged RHBDL4 in Hek293T cells at its C-terminus with a single FLAG-tag using CRISPR/Cas12-mediated gene editing ( Figure S5A-B) (Fueller et al, 2019). Hek293T cells expressing FLAG-tagged RHBDL4 were grown in medium supplemented with 'heavy' labeled amino acids whereas the parenteral Hek293T cells were cultured in normal medium.…”

“…Primers used for validation of Erlin2 knockout cells were: 5'-CTTGAGCAACGGCTGTATCC-3' and 5'-AATCACCACCCATGGCATCAT-3' leading to a 610 bp amplicon. Generation of chromosomally tagged RHBDL4-FLAG Hek293T cells with a single FLAG before the stop codon in the last exon by using CRISPR/Cas12 mediated gene editing has been described before (Fueller et al, 2019). Primers used for validation were: 5'-TTATGGAGCACGATGGAAGGAA-3' and 5'-GAGATGGGAGCGTGGAAACT-3', leading to a 634 bp amplicon.…”

“…Large, macroscopic aggregates cannot be targeted to the ERAD pathway and become subject for lysosomal degradation routes (not shown). (A) Outline of the applied tagging strategy of RHBDL4 (referred to also by its gene name Rhbdd1) according to (Fueller et al, 2019). Figure 6B normalized to wild type (wt) fibrinogen g-chain (means ± SEM, n=3).…”

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

“…Yet we needed to overcome the challenge of antibodies being often not sensitive enough detect subtle changes in protein expression. We therefore endogenously tagged both STT3 subunits at their C-terminus with a triple FLAG-tag using CRISPR/Cas12-mediated gene editing (Fueller et al, 2019), allowing for sensitive western blot detection ( Figure S2A and B). Indeed, as had been previously observed, knockdown of OST4 leads to a subtle reduction of the steady-state level of STT3A ( Figure 2A) (Dumax-Vorzet et al, 2013).…”

“…Generation of STT3A-FLAG and STT3B-FLAG Hek293T cells with a triple FLAG-tag before the stop codon in the last exon of the respective gene by using CRISPR/Cas12 mediated gene editing has been described before (Fueller et al, 2019). The cells used in this study where generated with the previously described Ctag-STT3A-LbCpf1.rev2 and Ctag-STT3B-LbCpf1.rev2 primer, respectively (Fueller et al, 2019).…”