PreMA TALEN reagent can be used to recapitulate previously reported loss-of-chrd-function phenotype in 1 dpf F0, injected larvae.

A. Top–Wildtype chrd sequence with TALEN binding sites annotated in teal. The dotted red boxes are MH arms predicted to be used most frequently. Raw sequence alignment of the whole PCR amplicon demonstrates that the majority of reads are the expected 7 bp deletion allele. Bottom–summary data from subcloning analyses. 50% of the mutant allele recovered were of the predicted MH allele. B. Previously reported chrd loss-of-function phenotype was successfully recapitulated using this TALEN pair. Phenotype severity was graded by the degree of Intermediate-Cell-Mass expansion in the tail and by the reduced head size by 1 dpf. Box plot demonstrating phenotypic penetrance is provided with each experiment denoted by a unique marker shape. N = 3 biological and technical replicates. At least 29 injected animals were scored in each experiment.

PreMA sgRNA against tyr can be used to recapitulate loss-of-melanophore phenotype in 2 dpf, injected F0 larvae.

A. Top–Wildtype tyr sequence with the #2 sgRNA target site annotated in green. The dotted red boxes are MH arms predicted to be used most frequently. Raw sequence alignment of the whole PCR amplicon demonstrates that the majority of reads are the expected 4 bp deletion allele. Bottom–summary data from subcloning analyses. 88% of the mutant allele recovered were of the predicted MH allele. B. Previously reported tyr loss-of-function phenotype was successfully recapitulated using this CRISPR-Cas9. Phenotype severity was graded by the loss of retinal pigmentation. Partial loss of retinal pigmentation was considered a Weak phenotype, whereas complete loss of pigmentation in one or both eyes were considered Moderate and Severe phenotypes, respectively. Box plot demonstrating phenotypic penetrance is provided with each experiment denoted by a unique marker shape. N = 3 biological and technical replicates. At least 12 injected animals were scored in each experiment.

Prospectively designed PreMA reagent against tdgf1 can be used to reproduce gross developmental defect in 1 dpf, injected F0 larvae.

A. Top–Wildtype tdgf1 sequence with sgRNA target site annotated in orange. The dotted red boxes are MH arms predicted to be used most frequently. Raw sequence alignment of the whole PCR amplicon demonstrates that the majority of reads are the expected 4 bp deletion allele. Bottom–summary data from subcloning analyses. 72% of the mutant allele recovered were of the predicted MH allele. B. Previously reported tdgf1 loss-of-function phenotype was successfully recapitulated using this CRISPR-Cas9. Phenotype severity was graded by the “pinhead” morphology and cyclopia. Pinhead morphology alone was classified as Weak, whereas Moderate and Severe phenotypes also presented with varying degrees of cyclopia judged by the distance of forebrain protrusion. In the Severe class, the forebrain does not separate the eyes, and they are fused together. Box plot demonstrating phenotypic penetrance is provided with each experiment denoted by a unique marker shape. N = 4 with 3 biological and 4 technical replicates. At least 42 injected animals were scored in each experiment.

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PreMA reagent can be used for in-frame gene alteration.

A. Top–Wildtype ttn.2 sequence with sgRNA target site annotated in red. The dotted red boxes are MH arms predicted to be used most frequently. Raw sequence alignment of the whole PCR amplicon demonstrates that the majority of reads are the expected 12 bp deletion allele. Bottom–summary data from subcloning analyses. 73% of the mutant allele recovered were of the predicted MH allele. B. 2 dpf zebrafish larvae injected with ttn.2 #2 sgRNA RNP (300 pg sgRNA + 660 pg Cas9) grossly appear normal with the exception of mild cardiac edema. Median penetrance was 50%. N = 3 biological and technical replicates. At least 9 injected animals were scored in each experiment.