HRM analysis reveals low frequency of off-target cleavage by the CRISPR system.

<p>Using the CRISPR Design (<a href="http://crispr.mit.edu/" target="_blank">http://crispr.mit.edu/</a>) web tool we identified the most likely 4 off-target sites in the zebrafish genome for 5 different guides with different overall scores (in parenthesis next to gene name). The sequences are depicted with the PAM site in green and the nucleotide different from the guide sequence in red. To discern SNP's within the Oft amplicons we performed HRM analysis of genomic DNA from 24 wild-type adults. The HRM positive frequency within genomic DNA obtained from 24 G0 embryos injected with the guide/Cas9 RNAs is displayed. Also the HRM positive frequency within genomic DNA obtained from 12 F1 embryos derived from 4 different G0's is displayed. Select HRM curves that display unique features are displayed for G0 embryos as well as F1 embryos. For Ptgs1 Oft 1 is displayed to depict no off-target hits. For Esco2 Oft 3 is displayed to depict a common SNP derived from the wild-type AB. For Wapal1 Oft 1 is displayed to depict no off-target hits. For P107 Oft 2 is displayed to: 1) depict a SNP found in wild-type AB strain, G0 and F1 animals; and 2) novel CRISPR derived off-target hits in some of the F1 progeny (orange and green curves). For Bub1bb, Oft 1 is displayed to depict a low frequency off-target hit (red curve) in the F1 progeny. * in frequencies denotes that SNP curves were not considered a HRM positive.