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. 2017 Mar 7;7:43733. doi: 10.1038/srep43733

Figure 3. Design of CRISPR/Cas9 targeting eEF1A1b and establishment of the eEF1A1b mutation line.

Figure 3

(a,b) Disruption of tilapia eEF1A1b by CRISPR/Cas9. Gene structure of eEF1A1b showing the target site and the BsajI restriction site. 300 ng/μl of Cas9 mRNA and 150 ng/μl of gRNA were co-injected into one-cell stage embryos. At 72 hours after injection, 20 embryos were randomly selected and pooled to extract their genomic DNA for PCR amplification. The indels were confirmed by two assays, restriction enzyme digestion and Sanger sequencing. The Cas9 mRNA and gRNA were added as indicated. Clear undigested band was detected in embryos injected with both Cas9 mRNA and gRNA compared with the control. (c) Schematic diagram showing the breeding plan of F0 to F1 fish. (d) eEF1A1b+/− F1 generation detected by restriction enzyme digestion. (e) Sanger sequencing results from the uncleaved bands were listed. Deletions are marked by dashes, and the PAM is marked in light orange. Numbers to the right of the sequences indicate the loss or gain of bases for each allele, with the number of bases inserted (+) or deleted (−) indicated in parentheses. WT, wild type.