CRISPR/Cas9-induced shank3b mutant zebrafish display autism-like behaviors

Abstract

Background: Human genetic and genomic studies have supported a strong causal role of SHANK3 deficiency in autism spectrum disorder (ASD). However, the molecular mechanism underlying SHANK3 deficiency resulting in ASD is not fully understood. Recently, the zebrafish has become an attractive organism to model ASD because of its high efficiency of genetic manipulation and robust behavioral phenotypes. The orthologous gene to human SHANK3 is duplicated in the zebrafish genome and has two homologs, shank3a and shank3b. Previous studies have reported shank3 morphants in zebrafish using the morpholino method. Here, we report the generation and characterization of shank3b mutant zebrafish in larval and adult stages using the CRISPR/Cas9 genome editing technique.

Methods: CRISPR/Cas9 was applied to generate a shank3b loss-of-function mutation (shank3b^-/- ) in zebrafish. A series of morphological measurements, behavioral tests, and molecular analyses were performed to systematically characterize the behavioral and molecular changes in shank3b mutant zebrafish.

Results: shank3b^-/- zebrafish exhibited abnormal morphology in early development. They showed reduced locomotor activity both as larvae and adults, reduced social interaction and time spent near conspecifics, and significant repetitive swimming behaviors. Additionally, the levels of both postsynaptic homer1 and presynaptic synaptophysin were significantly reduced in the adult brain of shank3b-deficient zebrafish.

Conclusions: We generated the first inheritable shank3b mutant zebrafish model using CRISPR/Cas9 gene editing approach. shank3b^-/- zebrafish displayed robust autism-like behaviors and altered levels of the synaptic proteins homer1 and synaptophysin. The versatility of zebrafish as a model for studying neurodevelopment and conducting drug screening will likely have a significant contribution to future studies of human SHANK3 function and ASD.

Keywords: ASD; Animal model; CRISPR/Cas9; Social behavior; Zebrafish; shank3.

Fig. 1

Generation of shank3b mutation in zebrafish by CRISPR-Cas9 gene editing. a Structure of zebrafish shank3b 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. Exon 2 is the target for CRISPR/Cas9 gene editing in zebrafish shank3b. The CRISPR/Cas9-induced mutation (5-base deletion and 13-base insertion) in shank3b is shown in annotated shank3b mutant sequences. The nucleotides in red are inserted sequences and the green highlighted “-” are deleted nucleotides. b Reduced expression of shank3b mRNA in the brain of shank3b^+/+ and shank3b^−/− adult (6 mpf) male zebrafish analyzed by RT-qPCR. c The expression of shank3a mRNA in the brain of shank3b^+/+ and shank3b^−/− adult (6 mpf) male zebrafish was not affected. Data are shown as mean ± SEM; ***p < 0.001

Fig. 2

Morphological characteristics of shank3b^−/− larvae and adult zebrafish. a–b Abnormal morphological changes in shank3b^−/− and shank3b^+/− larvae at ~ 1 dpf, including severe developmental delay, eye melanin reduction (blue arrow), and tail bending (red arrow) (+/+, N = 60; +/−, N = 50; −/−, N = 50). c–d Normal morphology and body length of shank3b^+/+, shank3b^+/−, and shank3b^−/− larvae at 3 dpf (c) and adults (6 mpf, male) (d) (N = 20 for each genotype). e–f Significantly enlarged brain size (e) but normal brain weight (f) in adult male shank3b^−/− (6 mpf) compared to WT zebrafish (N = 30 for each genotype). *p < 0.05

Fig. 3

shank3b^−/− larvae displayed impaired locomotion activity. a–c Spontaneous activity of shank3b^+/+, shank3b^+/−, and shank3b^−/− larvae was significantly reduced at 5 and 7 dpf, but not at 2 dpf. The X axis shows the intensity scale of the activity and Y axis shows the normalized activity frequency traveled by larvae in 1-min bin on each intensity scale (N = 24 for each genotype). d–f’ Light/dark test of shank3b^+/+, shank3b^+/−, and shank3b^−/− larvae at 5 and 7 dpf. The activity was recorded during 30 min of light (L0) and three 5-min light/dark intervals (D1/L1, D2/L2, and D3/L3) (d). The average distance moved within each 1-min bin under either light or dark conditions is plotted. Experiments were performed at 5 dpf (e and e’) and 7 dpf (f and f’). The vertical axis shows the normalized distance (millimeters) traveled by larvae in each 1-min bin. Data are shown as mean ± SEM (N = 24 for each genotype); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

shank3b^−/− adult zebrafish displayed reduced and repetitive locomotion activity in the open-field test. a Schematic diagram of the open-field test and thigmotaxis test of adult male zebrafish. In the analysis of thigmotaxis test, the area of the peripheral zone is equal to the center zone (dotted line). b–c shank3b^−/− zebrafish at 3.5 mpf showed significantly reduced velocity in the total 60 min period (b) and velocity per larva (c) in the open field (N = 13 for each group). d Representative traces of individual shank3b^+/+ or shank3^−/− zebrafish in the thigmotaxis test. e Ratio for the time spent and distance traveled (periphery divided by the total zone) over 30 min in adult male zebrafish (3.5 mpf). N = 13 for each group. f–g Representative trace of different types of stereotyped behaviors of shank3b^−/− adult male zebrafish (3.5 mpf). shank3b^−/− zebrafish had a significantly higher proportion of figure “8” and big circling movements than shank3b^+/+. N = 13 for each group. Data are shown as mean ± SEM; **p < 0.01, ****p < 0.0001

Fig. 5

shank3b^−/− zebrafish displayed social interaction defect. a–b Schematic of shoaling test (a) and significantly increased inter-fish distance of adult male shank3b^−/− zebrafish (3.5 mpf) (b). N = 18 for each group. c–f Schematic of social preference test of adult male zebrafish (3.5 mpf) (c). Heat map (d) shows that shank3b^+/+ zebrafish displayed significant higher frequency near a group of zebrafish than shank3b^−/− zebrafish. Time ratio (e) and distance ratio (f) in the conspecific sector were significantly reduced in shank3b^−/− zebrafish compared to shank3b^+/+ zebrafish. N = 16 for each group. g–h Schematic of kin recognition and preference test of adult male zebrafish (3.5 mpf) (g) and significantly reduced ratio of kin zone entering in shank3b^−/− zebrafish compared to shank3^+/+ zebrafish (h). N = 10 for each group. Data are presented as mean ± SEM; ****p < 0.0001

Fig. 6

shank3b deficiency altered the neurodevelopment in larvae. a–c Reduced RFP staining in shank3b^−/− larvae compared to shank3b^+/+ larvae (1 dpf, 2 dpf, and 3 dpf) using Huc: RFP transgene line zebrafish. The difference is the most prominent at 1 dpf. Scale bar, 100 μm. a’–c’ RT-qPCR results of RFP expressions from (a–c) larvae. N = 8 for each group. Data are presented as mean ± SEM; *p < 0.05, ****p < 0.0001

Fig. 7

shank3b deficiency resulted in the reduction of post- and presynaptic proteins in adult zebrafish brain. a Quantitative immunoblot blot analysis showed that the postsynaptic protein homer1 was significantly decreased (27% of shank3b^+/+) in the shank3b^−/− male zebrafish brain relative to shank3b^+/+ zebrafish (3.5 mpf, N = 3 for each group). b The expression of presynaptic synaptophysin protein was markedly reduced in shank3b^−/− male zebrafish brain compared with that of shank3b^+/+ zebrafish (3.5 mpf, 49% of shank3b^+/+). N = 3 for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01