CRISPR/Cas9 Homology Arm Cloning in Zebrafish

CRISPR/Cas9 target sites in exons of zebrafish genes were identified using CRISPRScan (http://www.crisprscan.org/; Moreno-Mateos et al., 2015 (link)). 5’ and 3’ homology arms of specified length directly flanking a genomic targeted double strand break were generated by annealing two complementary oligonucleotides. The double stranded 5’ and 3’ homology arms with appropriate overhangs were cloned into the pGTag vector BfuAI and BspQI sites, respectively, flanking the cargo. A three-nucleotide buffer sequence lacking homology to the genomic target site was engineered between the donor UgRNA PAM and the 5’ end of the homology arms. This was done in case the UgRNA PAM sequence was complementary to the nucleotides located 5’ to the start of the homology arm, which would increase the 24 or 48 bp homology arm length. Maps for the pGTag vectors and an open source protocol for cloning the homology arms are available at http://genesculpt.org/gtaghd/. The pGTag vectors are available through Addgene (https://www.addgene.org/kits/essner-geneweld/).
To generate 1 kb homology arms for zebrafish cx43.4 and esama, ~2 kb of genomic DNA surrounding the CRISPR target site was PCR amplified from adult WIK finclips using the proofreading enzyme KOD (EMD Millipore), and then sequenced to identify polymorphisms. Primers were designed to sit 1032 bp up and down stream of the cut site according to the Ensemble.org reference genome V11. Primers also contain either BfuAI and BspQI recognition sequences to make the appropriate overhangs for Golden Gate cloning into a pGTag vector or sequence for Gibson cloning into a pGTag vector. PCR was performed with the proofreading polymerase KOD and using genomic DNA from animals homozygous for the most common polymorphisms was used as template. The products were then Topo Blunt (Thermo Fisher Scientific) cloned for sequencing. The homology arms were Golden Gate or Gibson cloned into a pGTag vector containing the same cassette as the previous injections for the target locus. pGTag vectors with 1 kb homology arm vectors were injected into embryos from adults with the matching genomic sequence. Supplementary file 1 Table S5 lists the sequences of all homology arms, sgRNA target sites, and spacers. For each locus injections were done in triplicate, and for those targeting the locus with 1 kb homology arms the following controls were also performed; plasmid only, plasmid with universal gRNA and without genomic gRNA, and plasmid linearized in vitro with genomic gRNA.