Figure 1

CRISPR/Cas9-mediated reporter knock-in at the Actb locus in mouse haploid ESCs.

(a) Experimental scheme to establish haploid reporter ESC lines in culture. (b) Schematic illustration of the strategy to insert a P2A-Venus reporter cassette into the Actb locus. The upper diagram shows the Actb wild-type (WT) allele, which contains coding sequences (CDS) shown in black boxes and untranslated regions (UTR) shown in white boxes. The position of the single-guide RNA (sgRNA) target is indicated by a magenta arrowhead. The middle diagram shows the donor vector that contains the left and right homologous arms (HA-L and HA-R) and the Venus and neomycin resistance gene (neoR) driven by the PGK promoter. The bottom diagram represents the knock-in allele. Magenta arrows are PCR primers used to check the knock-in allele. (c) FACS analysis of Venus fluorescence after knock-in of the reporter cassette to the Actb locus. FACS analysis was performed 9 days after co-transfection of the donor vectors and the pX330 plasmids carrying a sgRNA targeting Actb (top) or Sox1 (bottom, Negative control) in haploid ESCs. (d) Bright field (BF) and fluorescence images of a Hap-AV cell line. A representative line (line No. 1) is shown. Scale bar, 200 μm. (e) Characterisation of the Hap-AV cell line using FACS. FACS analysis of the DNA content (left) and Venus fluorescence (right) are shown. (f) Targeted knock-in of the reporter cassette detected by genomic PCR in the Hap-AV lines. Representative 6 Hap-AV lines (No. 1–6) and their parental haploid ESC line (phES) are shown in the cropped gel images. Full length gels are presented in Supplementary Fig. S4. Primers and expected fragment sizes of each PCR amplicon; Actb-5’ = primer 1 and 2 (889 bp), Actb-3’ = primer 3 and 4 (800 bp), WT = primer 1 and 4 (1336 bp). (g) Sequence of the knock-in allele in the Hap-AV cells. The sequence of the WT allele is indicated at the bottom as a reference. PAM, protospacer adjacent motif.