Abstract
Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His6FLAG, His6-Biotin-TEV-RGSHis6, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.
Highlights
Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions
CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals
The linearized DNA fragment from the precise integration into target chromosome (PITCh) vector is integrated into the genome through short microhomologies via microhomology-mediated end joining (MMEJ)-mediated repair (Fig. 1A) [7]
Summary
The MMEJ-mediated PITCh tagging system utilizes the pX330 vector, which expresses the Cas nuclease and two gRNAs. MMEJ-mediated PITCh tagging is very efficient, but currently available systems are limited to modifications with GFP at the C terminus of endogenous proteins [6, 7]. We redesigned and optimized the MMEJ-based tagging approach for N-terminal tagging and expanded the repertoire of tags (Fig. 1B) This required rearranging the location of the GFP and the flanking microhomology sequence and addition of an additional 2A peptide. To test the efficiency of our approach, we designed a gRNA that targets the N terminus of the PP2AC ORF to insert the PITCh cassette containing GFP, PuroR, and 3ϫHA tag, all separated by 2A self-cleaving peptides. We transiently transfected 293T cells with pX330X2-PP2Ac-PITCh, which expresses the Cas nuclease and the two gRNAs that target the PITCh cassette for release of the repair fragment and the genomic sequence encoding the PP2Ac N terminus, respectively (Fig. 1A). This appears to be a problem caused by the HA tag only because no such degradation products were seen when other tags were fused to the PP2Ac N terminus (Fig. 2C)
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