Genotyping of F0 CRISPR-edited larvae using sanger sequencing and fluorescent in situ hybridization (FISH).

<p>(A, E, I) The gene structures for <i>mif</i>, <i>cd74a</i>, and <i>cd74b</i>. The sgRNAs for each gene are represented along with the general locations within each respective gene. The green text highlights the PAM sequences. The text in the parenthesis refers to the ensembl genome browser accession numbers, and the information that was entered into CHOP-CHOP to design sgRNAs. (B, F, J) FISH staining of uninjected vs. <i>mif</i>, <i>cd74a</i>, and <i>cd74b</i> CRISPR larvae, respectively. (C, G, K) The comparison CRISPR editing for 2 cut sites for <i>mif</i>, <i>cd74a</i>, and <i>cd74b</i>. Magenta represents cutting efficiencies greater than 50%, and black represent less than 50% cutting efficiency as predicted by TIDE or ICE analysis. R-squared values represent the sequence alignment between control and CRISPR-edited samples. (D, H, L) Representative signal traces comparing control to CRISPR edited larvae upstream of the PAM site for <i>mif</i> (B), <i>cd74a</i> (F), and <i>cd74b</i> (J). Scale bars = 50 μm. The data underlying the pie charts in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002194#pbio.3002194.s017" target="_blank">S2 Data</a>.</p> <p>(PDF)</p>