Figure 2 | Scientific Reports

Figure 2

From: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

Figure 2

Rapidly selecting most effective sgRNAs by single blastocyst genotyping.

(A) Overview and timeline of the experiment. Beginning with experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. (B) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red and the PAM sequence is labeled in purple. Deleted bases are marked with colons and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.

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