CRISPR-Cas9 F0 Crispant Knockout of slc24a5 in Zebrafish Using Multiplexed gRNAs with Heteroduplex Mobility and Pigmentation Scoring

AB embryos from >=3 pooled clutches are randomized into: uninjected, Cas9-only (no gRNA), scrambled-gRNA RNP, and slc24a5 4-guide RNP. n>=50 embryos/group/replicate, 3 independent replicates on separate days. Pigmentation is scored blinded at 72 hpf using a 0-3 ordinal rubric. A subset (n=24 individual larvae) per edited group is genotyped by HMA and a further n=10 by Sanger + ICE deconvolution to estimate indel spectra. Mosaicism is assessed per-individual.

BSL-1 under IACUC. Cas9 RNPs are non-infectious and low-hazard; handle with gloves. NaOH lysis is caustic — wear eye protection. Acrylamide (for PAGE) is a neurotoxin — use pre-cast gels or wear gloves/mask when handling powder; polymerized gels are lower risk but treat as hazardous waste. SYBR Safe and MS-222 handled with gloves. UV transilluminator: wear face shield/UV-blocking goggles. Euthanize per IACUC. Dispose chemical and biological waste through institutional streams.

Negative controls: uninjected and Cas9-only (no gRNA) confirm that pigmentation loss requires gRNA-directed cutting and that Cas9 alone is non-toxic. Specificity control: scrambled-gRNA RNP at matched concentration controls for nonspecific RNP toxicity. Molecular positive control: a previously validated gRNA against a different visible locus (e.g., tyr) run in parallel confirms RNP activity on the day. HMA includes a known wild-type homoduplex lane and a known edited heteroduplex lane as migration references. Biological reference: comparison to the golden (slc24a5) mutant.

slc24a5 4-guide crispants should show clear hypopigmentation in >70% of larvae (scores 2-3), with HMA showing prominent heteroduplex bands in >80% of genotyped individuals and ICE-estimated editing >80% with frameshift fraction >50%. Cas9-only, scrambled, and uninjected groups should show normal dark pigmentation (score 0-1) and homoduplex-only HMA. Mortality should be <20%. Phenotype-genotype concordance (pigment score vs % editing) should be strong (Spearman rho > 0.6).

• AB wild-type zebrafish embryos
• 4 synthetic sgRNAs targeting early exons of slc24a5 (Synthego/IDT), each tiling distinct PAM sites, 100 uM stocks
• Scrambled control sgRNA
• S. pyogenes Cas9-NLS protein (IDT/NEB), 10 ug/uL
• Cas9 buffer (20 mM HEPES, 150 mM KCl, pH 7.5)
• 0.05% phenol red tracer
• E3 medium + methylene blue
• DNA extraction: 50 mM NaOH lysis + 1 M Tris-HCl pH 8 neutralization, or HotSHOT
• Locus-flanking PCR primers (amplicon ~200-300 bp spanning all cut sites where possible, or per-guide)
• Taq polymerase, dNTPs
• 15% non-denaturing polyacrylamide (PAGE) or 4% MetaPhor agarose for HMA
• 100 bp ladder, SYBR Safe

To establish a robust 4-guide multiplexed CRISPR-Cas9 RNP injection workflow producing near-complete F0 loss-of-function (crispants) using slc24a5 (visible pigmentation reporter), with molecular verification by heteroduplex mobility assay and ICE indel deconvolution for rapid gene-function screening without breeding to homozygosity.

1. Assemble RNP on ice: combine each sgRNA (final ~1 fmol/nL ~ 300 ng/uL pooled) with Cas9 protein (final ~5 fmol/nL ~ 800 ng/uL) in Cas9 buffer; incubate 37 C for 5 min then keep on ice. Add phenol red to 0.05%.
2. Calibrate needle to deliver 1 nL.
3. Inject 1 nL of RNP directly into the cell (not yolk) at the 1-cell stage for maximal early editing.
4. Raise in E3 at 28.5 C; cull dead embryos at 6 and 24 hpf; record mortality.
5. At 72 hpf, anesthetize and score pigmentation blinded (0 = WT dark; 3 = fully golden/hypopigmented). Image representatives.
6. Lyse individual larvae (HotSHOT), PCR-amplify each target region.
7. For HMA: denature/reanneal PCR products (95 C 5 min, ramp to 25 C), run on 15% PAGE; edited samples show retarded heteroduplex bands. Quantify.
8. Sanger-sequence a subset and deconvolve indels with ICE/TIDE to estimate % editing and frameshift fraction.

Independent: presence/identity of gRNAs (4-guide slc24a5 vs scrambled vs none) at fixed RNP concentration. Dependent: per-larva pigmentation score, % editing by HMA/ICE, and frameshift fraction. Controlled: injection stage (1-cell), injection site (cell), volume (1 nL), Cas9:gRNA molar ratio, incubation temperature, clutch source, and scorer blinding.

Co-injection of 4 multiplexed slc24a5-targeting gRNAs (each complexed with Cas9 protein) into 1-cell embryos will produce >80% biallelic disruption, yielding visibly reduced/golden melanophore pigmentation in >70% of injected larvae by 72 hpf, phenocopying the golden mutant, whereas a scrambled gRNA RNP produces normal pigmentation.

Pigmentation scores are tabulated per individual and summarized as median/IQR per group. HMA gels are densitometered in Fiji; the heteroduplex:total band ratio estimates editing. Sanger traces are deconvolved with ICE (Synthego) to yield % indels and frameshift %. Phenotype-genotype concordance is assessed per individual by correlating pigment score with % editing. Indel size distributions are plotted as histograms.

1. Low editing efficiency: verify sgRNA integrity (run on gel), increase RNP concentration, ensure injection into the cell not yolk, and confirm 37 C RNP assembly. 2) High mortality: reduce Cas9 concentration, filter RNP to remove aggregates, and inject smaller volume. 3) No HMA heteroduplex despite phenotype: indels may be large/deletions removing a primer site — re-design flanking primers wider, confirm by Sanger. 4) Mosaic/partial pigmentation: expected for F0 crispants; inject earlier and use 4 guides to push toward biallelic LOF. 5) Multiple/smeared PCR bands: optimize annealing temp and primer specificity; large multiplex deletions can excise between guides.

Ordinal pigmentation scores are compared across groups by Kruskal-Wallis with Dunn's post hoc (alpha = 0.05). Percent-editing values (continuous) are compared by one-way ANOVA on arcsine-transformed proportions or Mann-Whitney. Phenotype-genotype concordance uses Spearman correlation. With 3 replicates of n>=50 for scoring and n>=24 genotyped/group, power exceeds 80% to detect a 40-point difference in median pigment score and a 30% difference in editing efficiency.