Deep Brain Photoreceptor (val-opsin) Gene Knockout Using CRISPR/Cas Affects Chorion Formation and Embryonic Hatching in the Zebrafish

Abstract

Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish.

Fig 1. A flowchart summarizing the generation of mutant zebrafish using the CRISPR/Cas system.

(1) microinjection of gRNAs and the Cas9 mRNA into embryos, (2) confirmation of mutation by DNA sequencing, (3) mating of an identified mutant (+/- or -/-, red) with a wild-type (+/+) partner, (4) phenotypic examination, (5) confirmation of germline transmission in the F1 generation, and (6) in silico protein prediction. According to the CRISPR/Cas concept, gRNA (brown line) and Cas9 nuclease (green circle) complex with genomic DNA (black double lines) at the gRNA target site near PAM (red dots on the double lines) to cleave dsDNA, leading to in-del mutations as a result of non-homologous end joining DNA repair. Abbreviation: CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated systems; dsDNA, double-stranded DNA; gRNA, guide RNA; PAM, proto-spacer adjacent motif.

Fig 2. Heritable mutations of the val-opsin isoform genes.

(a) Table for mutation rates at the valopa/b gRNA#1 and #2 sites in the F0 generations and the F1 offspring (*) derived from representative F0 valopa^rw03 and valopb^rw02 mutants. (b) Pie charts for mutation frequencies of individual F0 fish, i.e. valopa^rw01, valopa^rw02, valopa^rw03, and valopb^rw02 mutants, indicate different ratio of cloned DNAs with a wild type (WT), in-frame (IF), or frameshift (FS) genotype. Schematic drawings of the exons of the val-opsinA (c) and val-opsinB exons (d) indicate the DNA cleavage positions (dotted lines) with the guide RNA (gRNA)#1 and #2; and the PCR primer positions (yellow arrows) for genotyping. Details of the F1 offspring genotypes are provided below. The F1 genotypes contain deletions (dashes) and insertions (green letters) at the valopa/b gRNA#1 and #2 sites (black letters) near proto-spacer adjacent motif sequences (red letters). The numbers of deleted or inserted base pairs and resultant frameshift (FS) mutations are indicated. The numbers of mutants obtained with each genotype are shown in parentheses. Note that a F0 mutant with no detectable mutations at the valopb gRNA#2 site was used to produce F1 offspring (ND, not determined). N represents number of bacterial clones containing a target DNA.

Fig 3. Deduced amino acid sequences of the VAL-opsin proteins from representative mutants.

Alignments of partial peptide sequences indicating truncated peptide parts (grey highlights), targeted mutation sites (red bolts), and opsin-specific functional motifs such as the lysine K residue in the seventh transmembrane domain and the NPxxY(x)5,6F motif (green highlights) of VAL-opsinA (a) and VAL-opsinB (b). (c), Schematic drawing of the structures of wild-type VAL-opsin (left; transmembrane domains in green), and the truncated mutant VAL-opsins (right; transmembrane domains in red or blue) resulting from frameshift mutations (red lines) with premature stop codons. Accordingly, the VAL-opsin proteins resulting from frameshift mutations at the gRNA#1 site lack the K residue that binds to the light-absorbing chromophores (purple); and are rendered non-functional.

Fig 4. Abnormal chorion elevation in the eggs or embryos obtained from F0 female valopa/b mutants.

(a) Images of zebrafish eggs or embryos obtained at 4 hour-post-fertilization (hpf) from a female WT sibling and female mutants: valopa^rw01 (represented by A1), valopa^rw02 (A2), valopb^rw01 (B1), valopb^rw02 (B2), valopb^rw03 (B3). Upon spawning, chorion is elevated from the surface of the egg, and the blastodisc develops into embryonic cells in the created space (b). Arrows indicate the chorion surrounding the egg. Note that the eggs or embryos obtained from the valopa/b mutants exhibited no (single asterisk) or only partial (double asterisks) chorion elevation. Eggs with partial chorion elevation exhibited blastodisc expansion; although some develop into embryos, all eventually died (opaque appearance). (c and d) The mortality rates of the eggs or embryos and the hatching rates of embryos, respectively. Embryos from B1 and B2 mutants survived but mostly failed to hatch at 4 day-post-fertilization (dpf), in contrast to most embryos from the WT sibling hatched. Note that the association between the valop mutations in individual F0 parents and the increased number of dead eggs/embryos at 26 hpf and the decreased number of hatched embryos at 4 dpf was statistically significant (determined by Chi-Square X² values and p-values). Logistic Regression analysis showed that the observed phenotype is likely dependent on the targeted mutation (data in S1 File). N represents the number of eggs or embryos; and percentages shown in a single bar add up to a total of 100%. Abbreviation: bd, blastodisc; c, embryonic cells; Ch, chorion; y, yolk. Scale bar, 1 mm.

Fig 5. Late-hatching embryos obtained from a F0 male valopa mutant.

(a) Images of the zebrafish embryos obtained from a male WT sibling and a male mutant valopa^rw03 (represented by A3) at 0 day-post-fertilization (dpf), 4 dpf, and 6 dpf. (b and c) the mortality rates of the eggs or embryos and hatching rates of the embryos, respectively. Most embryos from the mutant had not hatched at 4 dpf, in contrast to most embryos from the WT sibling hatched. At 6 dpf, most of the unhatched embryos had died in the chorion. Genotyping of F1 adult offspring identified a mixture of WT and mutants. Note that the association between the valop mutations in individual F0 parents and the decreased number of hatched embryos at 4 dpf was statistically significant (determined by Chi-Square X² values and p-values). Logistic Regression analysis showed that the observed phenotype is likely dependent on the targeted mutation (data in S1 File). N represents the number of eggs or embryos; and percentages shown in a single bar add up to a total of 100%. Scale bar, 1 mm.

Fig 6. An overview of the parental effects of valop KO in the zebrafish.

Mating of F0 valopa/b mutants (male or female) with a WT partner resulted in F1 eggs or embryos exhibiting abnormalities (bold letters). Genotyping of the F1 offspring from valopa^rw03 and valopb^rw02 mutants indicate a mixture of WT and mutants and suggest the parental effects of valop KO. The parental-effect phenotypes observed in the current study is shared by the valopa and valopb mutants. This suggests that valopa and valopb co-expressing cells in the brain might be involved in the control of zebrafish reproduction. Abbreviation: c, embryonic cells; Ch, chorion; KO, knockout; y, yolk.