SplitGFP alleles enable visual selection of homozygous mutants.

a Schematic of actc1b gene structure, and location of the intron 2 and intron 4 guideRNA sites. b Schematic of intron 2 and intron 4 targeting vectors, FRT and FRT3 sites are indicated by triangles. c Schematic of Ti(actc1b^int2-mTagBFP2) and Ti(actc1b^int2-mTagBFP2-T2A-sGFP1-10) intron 2 targeted insertion lines. d Intron 4 targeted Ti(actc1b^int4-mTagBFP2-T2A-sGFP11x7) insertion line. e Example of successful targeted integration during the first cell division. Integration of the targeting vector into intron 2 of actc1b during the first cell division results in embryos with mTagBFP2 expression in one half of the ventral body plan. A white dotted line indicates embryo outline. The yellow dotted line indicates the ventral axis. In our experience, such embryos, when raised to adulthood and crossed, have always transmitted the successful integration event to their progeny. Ventral view of a 4 dpf embryo. f Brightfield and fluorescent images of wildtype, heterozygote, and homozygote embryos at 24 hpf. g 24 hpf embryos were generated by crossing the two actc1b^sGFP lines together. Heterozygous carriers display mTagBFP2 expression only while, in addition to mTagBFP2 expression, compound heterozygous mutants can be visually selected by GFP fluorescence. Experiments were repeated three independent times. Scale bar = 250 μm.

Site-specific integration of targeting vector into bag3.

a Schematic of bag3 gene structure, and location of the intron 2 guideRNA site. b Schematic of pSA0-mTagBFP2 targeting vector, FRT and FRT3 sites are indicated by triangles. c Schematic of Ti(bag3^int2-mTagBFP2) targeted insertion line. d Brightfield and fluorescent images of wildtype, heterozygote, and homozygote embryos at 24 hpf. Confocal images demonstrating loss of bag3 results in broken muscle fibres (yellow arrows) in homozygote mutants at 24 hpf after a 1 h 1% methylcellulose treatment. e mRNA expression analysis of bag3 demonstrates a complete loss of native bag3 transcript in homozygote mutants. f Fold change of mTagBFP2 mRNA levels in bag3^mTagBFP2 heterozygous and homozygous embryos compared to bag3 levels in wildtype siblings. Error bars represent SEM for n = 3 independent biological replicates, each consisting of a pooled sample of 16 or 19 embryos. rpl13 and ef1α were used as the reference genes. Source data are provided as a Source Data file. Statistical differences were determined using a two-tailed unpaired t-test.

Site-specific integration of targeting vector into tdgf1.

a Schematic of tdgf1 gene structure and location of the intron 3 guideRNA target site. b The pSA2-Gal4vp16/4xnrUAS-mTagBFP2 targeting vector, FRT and FRT3 sites are indicated by triangles. c Successful integration of the targeting plasmid to generate the Ti(tdgf1^int3-Gal4vp16/4xnrUAS-mTagBFP2) targeted insertion line. d Brightfield and fluorescent images of Ti(tdgf1^int3-Gal4vp16/4xnrUAS-mTagBFP2) targeted insertion line demonstrating loss of tdgf1 phenotypes in homozygote mutants at 24 hpf. mTagBFP2 fluorescence is false-coloured magenta. Scale bars = 250 μm. e, f mRNA expression analysis at 24 hpf demonstrates loss of tdgf1 in homozygote mutants (e) and the fold increase of mTagBFP2 expression levels compared to native tdgf1 from Gal4/UAS amplification (f). Error bars represent SEM for n = 3 independent biological replicates, each consisting of a pooled sample of 14 or 16 embryos. rpl13 and ef1α were used as the reference genes. Source data are provided as a Source Data file. Statistical differences were determined using a two-tailed unpaired t-test.

Site-specific integration of targeting vectors into vegfaa.

a Schematic of vegfaa gene structure, location of the intron 1 guideRNA target site. b The targeting vectors pSA2-Gal4vp16/4xnrUAS-mTagBFP2 and pSA2-Gal4vp16_synCoTC/4xnrUAS-mTagBFP2, FRT and FRT3 sites are indicated by triangles. c Successful integration of the targeting plasmid to generate the Ti(vegfaa^int1-Gal4vp16/4xnrUAS-mTagBFP2) hereafter referred to as vegfaa^afpUTR and Ti(vegfaa^int1-Gal4vp16_synCoTC/4xnrUAS-mTagBFP2) hereafter referred to as vegfaa^synCoTC targeted insertion lines. d, evegfaa targeted insertion lines demonstrating loss of vegfaa phenotypes in homozygote mutants at 48 hpf. dvegfaa^afpUTR transgenic insertion line. Scale bar 250 μm. evegfaa^synCoTC transgenic insertion line. mTagBFP2 fluorescence (magenta) in d and e was captured with the same imaging settings, demonstrating more robust expression levels in the synCoTC-containing line. Scale bar 250 μm. f mRNA levels at 24 hpf demonstrate the fold increase of mTagBFP2 levels compared to native vegfaa from Gal4/UAS amplification. Error bars represent SEM for n = 3 (vegfaa^afpUTR) or n = 4 (vegfaa^synCoTC) independent biological replicates, consisting of 17 or 18 (vegfaa^afpUTR) or 14 (vegfaa^synCoTC) pooled embryos. g Vascular labelling by the Tg(kdrl:EGFP) reporter demonstrates absence of the dorsal aorta (yellow arrow in wildtype) in mutants from both CRIMP lines, and branching defects of the trunk intersegmental vessels at 72 hpf. Scale bar 250 μm. h, i Both Ti(vegfaa^int1-Gal4vp16/4xnrUAS-mTagBFP2) (vegfaa^afpUTR) and Ti(vegfaa^int1-Gal4vp16_synCoTC/4xnrUAS-mTagBFP2) (vegfaa^synCoTC) CRIMP vegfaa mutants do not undergo genetic compensation. vegfaa and vegfab mRNA levels in vegfaa CRIMP mutants demonstrate complete loss of native vegfaa transcript, without a change in vegfab (h), demonstrating genetic compensation has not been induced. Error bars represent SEM for n = 3 (vegfaa^afpUTR) or n = 4 (vegfaa^synCoTC) independent biological replicates, consisting of 17 or 18 (vegfaa^afpUTR) or 14 (vegfaa^synCoTC) pooled embryos. rpl13 was used as the reference gene. ivegfaa^afpUTR and vegfaa^synCoTC CRIMP mutants have a reduction in the number of branching intracerebral central arteries (CtAs) similar to the reported non-compensating vegfaa promoterless mutant. vegfaa^{afpUTR−/−}n = 17, vegfaa^{synCoTC−/−}n = 19, wildtype n = 6. Error bars represent SEM. vegfaa^afpUTRp value = 0.0000002, vegfaa^synCoTCp value = 0.0000000003. Source data are provided as a Source Data file. Statistical differences were determined using a two-tailed unpaired t-test.

Schematic of CRIMPkit vector design.

CRIMPkit vectors contain a guideRNA target site (Hbait) for CRISPR/Cas9 mediated linearisation. The Hbait site is directly upstream of the 3’ region of β-actin intron 2, which contains the splice acceptor for exon-3. One or two bases have been added following the splice acceptor in pSA1 and pSA2 vectors respectively, to ensure the downstream reading frame is preserved. CRIMP vectors with a fluorophore after the splice acceptor a are designed for targeting genes with high expression levels. Vectors with Gal4vp16 after the splice acceptor and downstream UAS:fluorophore cassette b are for targeting genes with low expression levels. SA splice acceptor, afp pA Ocean pout anti-freeze protein 3’UTR until the poly-A sequence. ubc 5’UTR intron of ubiquitin C CoTC co-transcriptional cleavage. c Selection of correct CRIMP splicing vector. The reading frame of the last codon in the exon upstream of the intronic target site determines the correct vector to use in order to preserve the correct reading frame of the downstream coding sequence in a successfully targeted intron. A complete final codon will require a pSA0-vector, a codon missing one base requires a pSA1-vector, and a codon missing two bases requires a pSA2-vector to maintain the correct coding sequence. Black bases indicate the final codon of an exon upstream of an intronic target. Blue bases indicate the sequence incorporated in CRIMP vectors to maintain the reading frame after splicing.