Fig. 2

Homology-directed CRISPR-LbCpf1 knock-in of a pik3cd^E1017K allele. (A) Sequence of crRNA oligonucleotide, showing 5′ and 3′ extensions beyond the minimal sequence of repeat tail and target binding spacer. (B) Diagram illustrating the knock-in process. The CRISPR-LbCpf1 cleavage and knock-in is illustrated on the left and the resulting pik3cd^E1017K allele on the right. On the left, the ssODN donor sequence and crRNA spacer sequence are positioned alongside the pik3cd DNA sequence and translated amino acid sequence, with the CRISPR-LbCpf1 PAM highlighted in blue and the cleavage site shown as a red line. On the right, mutated DNA bases are shown in upper case, the E1017K coding change is highlighted in red, and the introduced ApoI cleavage site is indicated. The Ltest_1017 PCR primer is specific for the knock-in allele. (C) Map of the zebrafish pik3cd^E1017K locus. PCR primers are shown as magenta arrows; the mutation site is marked with a red asterisk; and positions are shown for the ssODN donor used for knock-in, the region confirmed by sequencing after knock-in and pik3cd exon 23. (D) Agarose electrophoresis gel with knock-in-specific test PCRs from 3 dpf G0 embryos. All the embryos previously injected with CRISPR-LbCpf1 and ssODN donor produced the expected 113 bp amplicon and sometimes also lower electrophoretic mobility products. PCRs from uninjected embryos did not amplify. DNA size marker is 100 bp ladder. (E) Agarose electrophoresis gel with knock-in-specific test PCRs from 3 dpf F1 embryos from an outcross of a G0 adult that had been injected with CRISPR-LbCpf1 and ssODN donor when an embryo. A pik3cd^E1017K/+ embryo produced the 113 bp amplicon; other embryos produced lower-mobility amplicons indicating aberrant knock-in events. DNA size marker is 100 bp ladder. (F) Agarose electrophoresis gel with PCRs spanning the knock-in locus to further test embryos that had given the expected 113 bp amplicon with test PCRs as in E. The pik3cd^E1017K/+ embryo indicated in E gave the expected 332 bp amplicon, but some other embryos produced lower-mobility amplicons indicating aberrant knock-in events despite having given the expected 113 bp amplicon with test PCRs as in E. (G) Agarose electrophoresis gel with Apo1-digested PCR amplicons from F2 adults from a pik3cd^E1017K/+ in-cross. The pik3cd^E1017K allele introduces an Apo1 site, allowing pik3cd^{E1017K/E1017K}, pik3cd^E1017K/+ and pik3cd^+/+ genotypes to be distinguished. DNA size marker is 100 bp ladder. (H) Sanger sequencing trace from a PCR amplicon across the knock-in locus of a pik3cd^{E1017K/E1017K} F2 adult. The mutated DNA bases are shown in upper case as in B.