Genome editing of slc25a46 gene in zebrafish using CRISPR/Cas9 system: (A) Schematic diagram of zebrafish slc25a46 gene with a frameshift in exon 8 indicated as a red box and a premature stop codon at amino acid position 238 (allele slc25a46^238s). The magenta arrows indicate five CRISPR guides injected together to create both F0 crispants and stable mutants. (B) Fragment analysis of the amplified CRISPR targeted region from the FAM-labeled PCR product (365 base pair amplicon) showing WT peak in control and multiple peaks indicating insertions and deletions (indels) in a representative slc25a46 F0 sample. X-axis represents base pair number; Y-axis represents signal intensity. (C) Sequence traces of wild-type (WT) and slc25a46^238s stable mutant zebrafish: exon 8, residues 223–238 indicating homozygous frameshift (in red); deletions are indicated with a triangle, insertion is underlined.

CRISPR/Cas9 targeting of slc25a46 gene in zebrafish leads to significant motoneuron axon disruption in slc25a46 F0 but not in slc25a46^238s zebrafish: (A) Confocal micrographs of 48 hpf zebrafish motoneurons stained with znp1 (cyan), whole mount, Z stack, lateral view captured above the yolk extension. Scale bar = 50 um. (B) Qualitative assessment of the primary motoneuron axon phenotypes: normal represent stereotypical “hook-like” axon path as in control images; disrupted–any number of abnormal motoneuron axons, such as axonal projections crossing into a nearby segment, truncated axons with projections not reaching back up to form the hook shape or aberrant hooks missing stereotypical branching pattern (indicated by white asterisks). P-values for comparisons between phenotypic penetrance in different genotypes are calculated by Fisher’s exact test (* for p<0.05; *** for p<0.001); n represents the number of individual larvae with observed motoneuron phenotype. Error bars represent SEM.

slc25a46^238s zebrafish are more resilient to slc25a46 morpholino (MO) injections than WT controls: (A) Confocal micrographs of 48 hpf zebrafish motoneurons stained with znp1 (cyan), whole mount, Z stack, lateral view captured above the yolk extension. Scale bar = 50 um. (B) Qualitative assessment of the motoneuron axon phenotypes: normal represent stereotypical “hook-like” axon path as in control images; disrupted–any number of abnormal motoneuron axons, such as axonal projections crossing into a nearby segment, truncated axons with projections not reaching back up to form the hook shape or aberrant hooks missing stereotypical branching pattern (indicated by white asterisks). P-values for comparisons of phenotypes between genotypes is calculated by Fisher’s exact test (* for p<0.05; *** for p<0.001). N represents the number of individual larvae with observed motoneuron phenotype. Error bars represent SEM. (C) Brightfield micrographs of 48 hpf zebrafish larvae, lateral view. Images represent the most commonly observed phenotype within a group. Scale bar = 0.5 mm. Red arrows indicate smaller eyes, eye coloboma, shorter trunk in phenotypically distinct zebrafish.

Slc25a46^238s zebrafish show a number of differentially expressed genes based on mRNA sequencing data: (A) Heat maps of all genes mapped to zebrafish genome comparing slc25a46^238s mutant to Cas9 control and slc25a46 F0 CRISPR; (B) Volcano plots showing differentially expressed genes between slc25a46^238s mutant versus Cas9 control or slc25a46 F0 compared to Slc25a46 F0 versus Cas9 control. Points above the red dashed line represent differentially expressed genes with p≤0.01; Red points represent chosen top genes of interest; (C) Upset plot showing intersections of upregulated and downregulated genes common (connected dots) and uniquely differentially expressed (individual dots) in slc25a46^238s mutant vs slc25a46 F0 and Cas9 control. The plot is based on the genes with p≤0.01.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.