There are four strategies being employed in the research community for measuring the efficacy of TALENs, CRISPR/Cas9 or Zinc Finger Nuclease-induced lesions in injected F0 embryos.The most popular and foolproof method is PCR amplification of the locus from pooled lysates of 5-10 embryos, followed by Topo-TA cloning and brute force sequencing of several dozen clones (colony PCR can help with this). The following three alternate methods can save time in the long run, which can be particularly useful for identifying adult zebrafish carriers, but they each require a time investment for optimization, and each can be sensitive to SNPs outside of the target.
- Loss of a restriction recognition site overlapping with or close to the target site
- Detection of increased mismatches with surveyor nuclease
- Detection of mismatches by high-resolution melting analysis (HRMA). The NICHD zebrafish core has expertise with this technique. Please contact Ben Feldman for advice.