Figure 2. Co-injection of Scr7 enhances the efficiency of precise genome editing in mouse embryos.
(a) Strategy for introduction of a sortase target motif at the stop codon of the mouse Kell locus and the Ig κ constant region. sgRNA guide sequences of the Kell 3’ exon and the Igκ constant region are indicated in green. The Kell 3’ exon and the Igκ constant region are capitalized. A PAM motif is indicated in bold. The targeting single-stranded (ss) DNA template [targeting template (ssDNA)] contains ~80 nt of homology flanking both sides of the DSB. In the case of Igκ, the PAM is deleted to avoid inducing DSBs on the targeting ssDNA template as shown by “^”. The insertion cassette, including sortase recognition motif and linker, is labeled in blue. (*: stop codons).
(b) A mixture of the CRISPR components (Cas9 mRNA, sgRNA and targeting template) was prepared, and 10 mM of Scr7 NHEJ inhibitor was then added to the mix (final concentration: 1mM). Fertilized zygotes were collected from oviducts of super-ovulated female mice, and the mixture was injected into the cytoplasm at the pronuclear stage. The injected zygotes were transferred at the 2-cell stage into the oviduct of pseudo-pregnant females.
(c) Genotyping results of the Kell-targeted blastocysts. Co-injection was conducted as in (b). The co-injected zygotes were cultured up to the blastocyst stage. The blastocysts were subjected to PCR using an LPETG-specific forward primer and a Kell-specific reverse primer. The PCR-positive blastocysts were counted as insertion positive. P value was calculated by Fischer's test in Prism6 software. (p=0.0012)
(d) Restriction fragment length polymorphism (RFLP) analysis results of the Kell-targeted E10 embryos. Upper: the untreated, original amplicons using Kell external primer, lower: the digested amplicon using a unique enzyme MboI to LPETG cassette, including a linker sequence and the LPETG sortase-targeting motif. Genotypes and sequencing results are shown as ‘Genotype’(+: wild-type allele,Δ: deletion mutant allele, di: insertion mutant allele) and ‘Observed indel’ at the bottom (minus: deletion; plus: insertion).
(e) Primer sets to detect insertion of the sortase motif. External primers were designed outside of the homology arms flanking the DSBs. Primer set #1: a sortase motif-specific forward primer, and external reverse primer targeting the Igκ constant region > 250 bp downstream of the DSB, primer set #2: a pair of external forward and reverse primers targeting the genomic region > 250 bp up- and down-stream of the DSB.
(f) Genotyping results of the Ig κ constant region-targeted E10 embryos. Upper: genotyping results using primer set #1 in Fig. 2e. The amplicon obtained using primer set #1 in figure 2e is shown as an arrow. (*: non-specific product) Lower: the amplicon obtained using primer set #2 in figure 2e. Genotypes and sequencing results are shown as ‘Genotype’(+: wild-type allele,Δ: deletion mutant allele, di: insertion mutant allele) and ‘Observed indel’ at the bottom (minus: deletion; plus: insertion). RFLP analysis by digestion with MboI is shown in the Supplementary Figure 5e.