A generic strategy for CRISPR-Cas9-mediated gene tagging

Abstract

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.

Figure 1. Approach for homology-independent gene tagging.

(a) Schematic representation: cells are transfected with Cas9, a gRNA specifying the desired locus in the human genome (here in exon 9 of the gene of interest) and a generic donor plasmid. The generic donor plasmid contains the tag of interest, flanked by two tia1l recognition sites, as well as a U6 promoter driving the expression of the tia1l gRNA. Consequently, the tag of interest will be released upon co-expression of Cas9 and spontaneously integrated at the site specified by the genomic gRNA. (b) HAP1 cells were transfected with expression plasmids for Cas9, the tia1l gRNA and the generic NanoLuc donor. For each gene under consideration, we chose two independent gene-specific gRNAs that were co-transfected. Genomic DNA was isolated from pools of cells 5 days post transfection and analysed by PCR. For this PCR, one constant primer binding to the NanoLuc cassette (5′-GGATCGGAGTTACGGACACC-3′) was combined with one variable primer for each gene of interest. HAP1 wild-type cells were included as a reference (lanes labelled with —). Numbers above each lane define the guide RNA identity, as specified in Fig. 2 and Table 1.

Figure 2. Gene tagging is efficient and precise.

Sanger sequencing data of clones described in Table 1 were analysed to identify the integration pattern. Dark-blue arrows indicate the directionality of the tia1l gRNA, light-blue arrows indicate the directionality of the genomic gRNA site. Red dots symbolize additional insertions or deletions identified by Sanger sequencing. Number on the left specify the clone ID.

Figure 3. Cell lines bearing NanoLuc integrations can be used to monitor changes in gene expression.

(a) HAP1 cells were stimulated with various cytokines as indicated (IFN-β, activin A and FGF1) for 4, 8 or 24 h at a final concentration of 50 ng ml⁻¹. RNA was isolated and analysed by qPCR for the following signature genes: IFIT1, DACT1 and EGR1. Error bars show the s.d. from three technical replicates. (b) Clonal cell lines bearing NanoLuc integrations in IFIT1, DACT1 and EGR1 (Table 2) were stimulated with IFN-β, activin A or FGF1 as indicated. Cell lines were collected after 24 h (for IFIT1 and DACT1) or after the indicated time points (for EGR1) and NanoLuc luciferase levels were measured. Error bars show the s.d. from six technical replicates.

Figure 4. Cell lines bearing TurboGFP can be used to monitor subcellular localization.

(a) HAP1 cells were transfected with expression plasmids for Cas9 and the generic TurboGFP donor that expresses the tia1l gRNA. In addition, we co-transfected one gRNA for each gene under consideration (LMNA, TERF1 and LAMP1). TurboGFP-positive cells were enriched by FACS. (b) Clonal cell lines bearing TurboGFP integrations in LMNA and TERF1 were fixed, stained with 4,6-diamidino-2-phenylindole (DAPI) and analysed by fluorescence microscopy.