Fig. 1

Generation of a floxed Pitx1 allele using Easi-CRISPR. a–d The Easi-CRISPR strategy. a The two parts of the CRISPR guideRNA (crRNA + tracrRNA) and Cas9 protein. Combining them generates a ctRNP complex. The term ctRNP used here was formerly known as a cloning-free CRISPR/Cas9 system [28]. b A long ssDNA donor derived from a floxed exon cassette (or knock-in cassette as in Fig. 3) is mixed with ctRNP(s) to obtain the final Easi-CRISPR reagent cocktail for zygote injection. c Injection of a floxed ssDNA donor with right and left ctRNPs into zygotes results in replacement of the target exon with the floxed exon. For targeted insertions (as in Fig. 3) only a single ctRNP is required. d Following microinjection of the Easi-CRISPR reagent cocktail, genotyping and sequencing are used to identify founders with correctly modified genomes. e, f The Pitx1 wild-type allele, the Pitx1 ssDNA donor designed to flox exon 2 and the final targeted allele. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. f Three genotyping PCRs and the primer combinations for these are indicated (5′ LoxP PCR, 5′ F + 5′ R primers; 3′ LoxP PCR, 3′ F + 3′ R primers; and full-length PCR, 5′ F + 3′ R primers). g Genotyping gel images from the ears of G0 offspring. The expected sizes of PCR amplicons (wild type (wt) or floxed) are indicated to the left of the gels. h Genotype interpretations are summarized below the gel image (M monoallelic, P partial insertion, N no insertion). Animals 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites in cis, while animals 2, 4, 9, and 10 contained only one LoxP site, due to partial insertion of the ssDNA cassette