Figure 1.

CRISPR/Cas9-Mediated Knockin of mCherry into COUP-TFII

(A) Location and sequence of single-guide RNAs (sgRNAs) 1 and 2 in the COUP-TFII locus.

(B) Schematic representation of the CRISPR/Cas9 plasmid-based targeting and dual-selection protocol.

(C) Schematic overview of wild-type (WT) COUP-TFII, COUP-TFII-mCherry targeting construct, and the resulting targeted (first allele) and WT COUP-TFII (second allele) alleles with forward (FWD) and reverse (RV) primer binding sites for screening.

(D) PCR screen to identify targeted clones: Upper panel: 3′ end screen and confirmation of excision of the blasticidin-resistance cassette in 1, 2, 4, 5, and 6, but not 3 (targeted, but blasticidin-resistance cassette still present). Middle panel: 5′ end screen shows that clones 1–3, and 5 and 6, were targeted at the 5′ end. Lower panel: screen to determine if heterozygous (clones 2, 3, 4, and 6) or homozygous (clones 1 and 5) mCherry knockin occurred.

(E) Schematic representation of the CRISPR/Cas9-mediated correction using a WT COUP-TFII single-stranded oligonucleotide (ssDNA oligo).

(F) Sequence corresponding to the 9 bp deletion in the COUP-TFII locus along with the annealing sites for sgRNA 3 designed to repair the deletion.

(G) Sanger sequencing confirming correction of the second COUP-TFII allele from the COUP-red clone after correction.

See also Figures S1 and S2.