Fig. 5

Fig. 5 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.