Fig. 3

Design of ssODN and screening strategy for knock-in of a point mutation in gba. A Schematic of gba genomic structure with coding region shaded in black (top panel), alignment of genomic sequence, sequence in our cohort of fish showing 2 polymorphisms (highlighted in blue) and ssODN design with direction of 36 bp and 91 bp homology arms (HA) marked. Amino acids coded by each triplet are marked above the genomic sequence, with the amino acid to be changed marked in red. The ssODN contains a modification to the PAM site (G > C, highlighted in red) which creates a SalI restriction site (marked by black rectangle), a G > A modification to change the SNP in our WT cohort back to the reference sequence (highlighted in green) and the desired point mutation A > C (highlighted in yellow). B A schematic of the strategy for combining fluorescent PCR and RFLP analysis to detect integration of ssODN. Top panel shows the 3-primer fluorescent PCR strategy with fluorophore denoted by blue star and restriction sites marked as RS. Middle panel shows the two possible outcomes in injected embryos with size of the PCR product and location of either 2 (insertion of ssODN) or 1 (no insertion of ssODN) restriction sites (marked in red). The bottom panel shows the expected fragment sizes after the enzymatic digestion of the PCR product. Since only the fluorescently labelled fragments will be detected, embryos with knock-in can be identified by the presence of a 111 bp fragment. Multiple peaks around the WT size of 235 bp are expected due to CRISPR-induced random indels. C Representative CRISPR-STAT plots of uninjected, sgRNA/Cas9, and sgRNA/Cas9 plus ssODN injected embryos before and after SalI digestion with X-axis showing the size of the peaks, Y-axis showing the peak height, and the size of the expected WT allele denoted by a green arrowhead. Successful integration following digest results in a peak at 111 bp (denoted by red arrowhead)