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Fig. 3 | Genome Biology

Fig. 3

From: Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Fig. 3

Fusion of P2A-FlpO to the 3′ end of Fgf8 using Easi-CRISPR. a How Easi-CRISPR is used to generate knock-in alleles. b The Fgf8 locus, ssDNA donor, and the resulting targeted insertion allele. c Genotyping of G0 offspring. Primer locations for 5′ and 3′ junction PCRs are shown, along with expected amplicon sizes. Founder 4 has a correctly targeted P2A-FlpO insertion, as indicated by the presence and size of both 5′ and 3′ junction amplicons. The gel on the right shows that PCR amplification of this founder’s DNA with primers flanking the Fgf8 insertion site produced only the mutant amplicon, indicating that it is a biallelic insertion. WT wild type, M 100-bp marker; kb 1-kb marker. d Sequencing of 5′ and 3′ junctions in founder 4. The guide RNA sequence (italics), along with the cut site, PAM sequence (in red), a few bases of flanking sequences (above) and sequence chromatograms showing correctly targeted 5′ and 3′ junctions are shown below

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