Generation of a zebrafish shank2b mutant using CRISPR/Cas9. (A) Structure of zebrafish shank2b gene and protein. The protein domains (ANK, ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, PSD-95/Discs large/ZO-1 domain; SAM, sterile alpha motif domain) are aligned to the corresponding exons. Diagram of target site on 10th exon of zebrafish shank2b genomic DNA and a 14 bp deletion mutation by CRISPR/Cas9 gene editing. Predicted structures of wild-type (WT) and shank2b mutant proteins in zebrafish. The 14 bp deletion resulted in a stop codon on 13th exon and premature termination before the PDZ and SAM domains. (B) Reduced expression of shank2b mRNA in the brain of shank2b−/− adult male zebrafish at 4.5 months post-fertilization (mpf) compared to WT fish, analyzed by RT-qPCR (WT n = 4, shank2b−/− n = 4, **** p < 0.0001, Student’s t test). Data are shown as mean ± SEM. (C) The expression of shank2a mRNA in the brain of WT and shank2b−/− adult (4.5 mpf) male zebrafish was not affected (WT n = 3, shank2b−/− n = 4, ns, p = 0.1957, Student’s t test). Data are shown as mean ± SEM.
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