A single short homology arm 5’ to the sgRNA target site in the noto gene targets integration in zebrafish embryos. (a) Schematic for noto homology arm and donor vector design. Bold letters show the noto sgRNA target sequence in the genome. This sgRNA target sequence was also used to target Cas9 cutting in the donor vector. Black bars represent the different homology arm lengths 12, 24, or 48 bp, used to target the 2A-tagRFP-CAAX donor vector into the noto exon 1 target site. PAM sequences are underlined. Red arrows indicate the Cas9 cut site 3 bp upstream of the PAM. The 3 nucleotide spacer lacking homology to the genome is represented by the lowercase sequence ‘aaa’ located in between the donor vector PAM and the 5’ end of the homology arm. (b) Targeting efficiency of noto exon1 2A-tagRFP-CAAX donor vectors containing a single 5’ homology arm of 12, 24, or 48 bp. Data represents mean +/- s.e.m. of 3 independent targeting experiments. p values calculated using two-tailed unpaired t-test. (c) Live confocal image of noto-2A-TagRFP-CAAX-SV40 targeted embryo showing specific RFP expression in the notochord. Scale bar, 100 μm. (d) Sanger sequencing of cloned 5’ junction fragments from RFP positive F0 embryos, aligned to the expected sequence from a precise integration event. Numerator represents correct clones, denominator represents total clones sequenced. Junctions are considered precise if the homology arm does not contain any mismatch and there are no insertions or deletions up- or downstream of the programmed homology.
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