(A) Diagram of the UFlip. The vector contains a floxed rox loxP lox2272 gene trap plus secondary marker loxP lox2272 rox cassette. The cassette is flanked by cloning sites for homology arms (HA) complementary to a genomic CRISPR target site, and universal gRNA sites (UgRNA) for in vivo liberation of the targeting cassette. (B) Gene ‘off’ alleles are generated by integration of the UFlip cassette into an intron in the active orientation, leading to transcription termination and splicing of the primary transcript in the mRFP gene trap. (C) Gene ‘on” alleles are generated by integration of the UFlip cassette into an intron in the passive orientation. This is driven by cloning the genomic 5’ homology arm downstream of the UFlip cassette, and cloning the genomic 3’ homology arm upstream of the UFlip cassette. Integration at the genomic CRISPR-Cas9 target site occurs in the opposite orientation. During transcription RNA polymerase reads through the integrated UFlip cassette, which is spliced out with the intron during processing of the primary transcript. (D) Cre-mediated recombination at an ‘off’ allele locks the cassette in the ‘on’ orientation. The first recombination occurs stochastically at either lox2272 or loxP sites. The diagram shows the intermediate that forms if the first recombination occurs at the lox2272 sites. (E) Cre-mediated recombination at an ‘on’ allele locks the cassette in the ‘off’ orientation. The first recombination occurs stochastically at either lox2272 or loxP sites. The diagram shows the intermediate that forms if the first recombination occurs at the lox2272 sites. BFP, blue fluorescent protein; gcry1, gamma crystallin 1 promoter; myl7, cardiac myosin light chain 7 promoter; 2 A, porcine teschvirus-1 2A peptide; mRFP, monomeric red fluorescent protein; pA, transcription termination and polyadenylation signal; SA, splice acceptor.

(A) rbbp4 gene model with sequence of the intron 4 reverse strand gRNA. Gel image of PCR amplicons surrounding the target site from 8 Cas9 plus gRNA injected and 1 uninjected (U) embryo. Amplicons from embryo #3 and the uninjected embryo were sequenced and analyzed with Synthego’s ICE software, and indicate 50% indel efficiency at the target site. Plot shows the range and percentage of indels present in the sequences. PAM sequence shown in bold and underlined. (B) rb1 gene model with sequence of the intron 6 reverse strand gRNA. Gel image of PCR amplicons surrounding the target site from eight embryos injected with Cas9 and the gRNA (1-8), and two uninjected embryos (U). Amplicons from embryo #1 and an uninjected embryo were sequenced and analyzed with Synthego’s ICE software, and indicate 95% indel efficiency at the target site. Plots show the range and percentage of indels present in the sequences. PAM sequences shown in bold and underlined. (C) 5’ and 3’ genomic-UFlip integration junctions were PCR amplified from F1 transgenic zebrafish fin clip genomic DNA. The PCR products were sequenced and aligned to the reference sequence expected for a precise integration at the genomic target site. Capitalized red nucleotides represent 48 bp homology arms. Lowercase green nucleotides represent random inserted sequences.

(A) Diagram illustrating Dre-mediated inversion of the rbbp4^off allele to the on orientation. Repeated inversion of the cassette will continue as long as Dre is present. The final allele is predicted to be in the inverted ‘on’ orientation at a frequency of 50%. (B) PCR junction analysis of 8 embryos from an F1 adult that had been injected with Dre mRNA at the one-cell stage. Three embryos positive for expression of the lens BFP secondary marker show the expected 5’ and 3’ junction PCR amplicons for the inverted rbbp4^on allele. (C) Sequence analysis confirms Dre-mediated inversion of the cassette from the ‘off’ to ‘on’ orientation in BRP+/RFP - embryos.

(A) Diagram of the rbbp4^off allele. (B) Plot of RT-qPCR results from wild type +/+ (n=3), heterozygous rbbp4^off/+(n=3), and homozygous rbbp4^off/off (n=3) larvae showing the relative level of rbbp4 mRNA transcript using reference gene rps6kb1b. Primer pairs were located in exons 4 and 5, or downstream exons 11 and 12. (C – E) Gross phenotype of rbbp4^Δ4/+ (C), rbbp4^off/Δ4 (D), and rbbp4^Δ4/Δ4 (E) 5 dpf larvae. Arrowhead in (D) points to overlap of rbbp4^off 2A-mRFP primary reporter and gcry1:BFP secondary reporter expression in the lens, which appears purple. (F – H) Caspase-3 and HuC/D labeling of sectioned head tissue from 2 dpf rbbp4^Δ4/+ (F) rbbp4^off/Δ4 (G) and rbbp4^Δ4/Δ4 (H) embryos. (I) Diagram of the rbbp4^on allele. (J) Plot of RT-qPCR results from wild type +/+ (n=3), heterozygous rbbp4^on/+ (n=3), and homozygous rbbp4^on/on (n=3) larvae showing the relative level of rbbp4 mRNA transcript using reference gene rps6kb1b. Primer pairs were located in exons 4 and 5, or downstream exons 11 and 12. (K, L) Gross phenotype of rbbp4^on/+ (K) and rbbp4^on/Δ4 (L) 5 dpf larvae. The rbbp4^on allele secondary marker gcry1:BFP expression is visible in the lens. Caspase-3 and HuC/D labeling of sectioned head tissue from 2 dpf rbbp4^Δ4/+ (M) and rbbp4^off/Δ4 (N) embryos. OT, optic tectum; R, retina; Th, thalamic region. Error bars represent mean ± s.e.m. Scale bars: 200 μm (C–E, K, L), 50 μm (F–H, M,N).

(A) Diagram of expected Cre mediated inversion of rbbp4^off to on orientation. (B) Gross morphological phenotype of microcephaly and microphthalmia in 5 dpf transheterozygous rbbp4^off/Δlarva. (C) Cre injected 5 dpf transheterozygous rbbp4^off/Δ4larva shows rescue of gross phenotype and loss of mRFP expression. (D) Activated caspase-3a labeling throughout midbrain and retina section from 2 dpf transheterozygous rbbp4^off/Δ4embryo. (E) Absence of activated caspase-3a labeling in midbrain and retina of 2 dpf transheterozygous rbbp4^off/Δ4embryo after Cre injection. (F) Quantification of caspase-3a labeling in control rbbp4^off/Δ4(n=3) and Cre injected rbbp4^off/Δ4 (n=3) midbrain (** p<0.01) and retina (**** p<0.0001). (G) Genomic DNA qPCR quantification of rbbp4^off original orientation 5’ and 3 junctions in control rbbp4^off/Δ4 (n=3) and Cre injected rbbp4^off/Δ4 (n=3). Cre injection reduced the level of rbbp4^off original orientation 5’ (>93%) and 3’ junctions (>93%). (H – J”) Activated caspase-3A and Cre labeling in sectioned head tissue from 2 dpf rbbp4^off/Δ4 (H-H"), ascl1b-2A-Cre; rbbp4^off/Δ4 (I-I"), and neurod1-2A-Cre; rbbp4^off/Δ4 (J-J") embryos. (K) Quantification of caspase-3a labeling in rbbp4^off/Δ4(n=3), ascl1b-2A-Cre; rbbp4^off/Δ4 (n=3) and neurod1-2A-Cre; rbbp4^off/Δ4 (n=3).rbbp4^off/Δ4vs. ascl1b-2A-Cre; rbbp4^off/Δ4 midbrain (n.s. p=0.3248) and retina (n.s. p=0.8153), and neurod1-2A-Cre; rbbp4^off/Δ4 midbrain (n.s. p=0.7794) and retina (n.s. p=0.9365). OT, optic tectum; R, retina; Th, thalamic region. Error bars represent mean ± s.e.m. with two-tailed t-test. Scale bars: 200 μm (B, C), 50 μm (D, E, H – J). 10 μm (H’ – J”).

(A) Diagram of the rb1^off allele. (B) Plot of RT-qPCR results from wild type +/+ (n=3), heterozygous rb1^off/+ (n=3), and homozygous rb1^off/off (n=3) larvae showing the relative level of rb1 mRNA transcript using reference gene rps6kb1b. Primer pairs were located in exons 6 and 8, or downstream exons 23 and 24. (C – E) Gross phenotype of wildtype +/+ (C), rb1^off/stop (D), and rb1^Δ7/stop (E) 5 dpf larvae. Arrowhead in D points to rb1^off allele gcry1:BFP secondary reporter expression in lens. Asterisk marks the rb1^stop allele mly7:GFP secondary reporter expression in heart. (F – H) pH3 and HuC/D labeling of sectioned head tissue from 5 dpf +/+ (F), rb1^off/stop (G), and rb1^Δ7/stop (H). (I) Diagram of the rb1^on allele. (J) Plot of RT-qPCR results from wild type +/+ (n=3), heterozygous rb1^on/+ (n=3), and homozygous rb1^on/on (n=3) larvae showing the relative level of rb1 mRNA transcript using reference gene rps6kb1b. Primer pairs were located in exons 6 and 8, or downstream exons 23 and 24. (K, L) Gross phenotype of wildtype +/+ (K), heterozygous rb1^on/+ (L) and transheterozygous rb1^on/stop (M) 5 dpf larvae. Arrowhead in L, M points to rb1^off allele gcry1:BFP secondary reporter expression in lens. Asterisk in M marks the rb1^stop allele mly7:GFP secondary reporter expression in heart. pH3 and HuC/D labeling of sectioned head tissue from 5 dpf +/+ (N), heterozygous rb1^on/+ (O) and transheterozygous rb1^on/stop (P).OT, optic tectum; R, retina; Th, thalamic region. Error bars represent mean ± s.e.m. Scale bars: 200 μm (C–E, K–M), 50 μm (F–H, N–P).