Generation of shank3a and shank3ab mutants in zebrafish by CRISPR-Cas9 gene editing. (A) Structures of zebrafish shank3a and shank3b (Liu et al., 2018) gene and protein domains (ANK, ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, PSD-95/discs large/ZO-1 domain; SAM, sterile alpha motif domain). Exon 9 is the target for CRISPR/Cas9 gene editing in zebrafish shank3a and exon 2 is the target for shank3b. The CRISPR/Cas9 induced frameshift mutations in shank3a (5-base deletion) and in shank3b (5-base deletion and 13-base insertion) which led to the truncation of the protein (Liu et al., 2018). (B) The mutations of shank3a and shank3b (Liu et al., 2018) were verified by Sanger sequencing. The predictions of protein spatial structures were both suggested that shank3a and shank3b proteins were likely to turn into truncated proteins (https://www.swissmodel.expasy.org/interactive/wUdwQQ/models/). (C) Reduced expression level of shank3a mRNA in the brain of shank3a– /– and shank3ab– /– adult (4 mpf) male zebrafish analyzed by RT-qPCR, while shank3b– /– was not affected. (D) Reduced expression level of shank3b mRNA in the brain of shank3b– /– and shank3ab– /– adult (4 mpf) male zebrafish analyzed by RT-qPCR, while shank3a– /– was not affected. Data are shown as mean ± SEM; **P < 0.01. ****P < 0.0001.
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