Figure 1
Analysis of mutagenesis efficiencies of CRISPR/Cas9 in the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM1) genomic gene of channel catfish (Ictalurus punctatus). (A) Identification of mutated TICAM1 gene sequences in channel catfish. For each sample, (a) half of the PCR product was resolved in a 2% agarose gel to detect the large deletion while (b) the other half was digested with Surveyor® enzyme so that (a) and (b) were from the same embryo. Samples 1–6 came from embryos that were microinjected with gRNA/Cas9 protein while lanes W represent wild type channel catfish that were full-sib to embryos 1–6. Embryos with a large deletion had short band(s) when compared to the wild type (750 bp) band. M indicates 1 kb plus DNA ladder (Invitrogen). Electrophoretic results were cropped from the original images shown in Supplementary Fig. S1. (B) Mutation rate ± standard error (SE) of channel catfish dead embryos and alive fingerlings microinjected with gRNA/Cas9 protein targeting TICAM 1 gene. Three dosages were microinjected into one-cell embryos (low dosage: 2.5 ng gRNA/7.5 ng Cas9, medium dosage: 5 ng gRNA/15 ng Cas9 and high dosage: 7.5 ng gRNA/22.5 ng Cas9). (C) CRISPR/Cas9 induced mutations in the TICAM1 gene of channel catfish. The wild-type channel catfish TICAM1 genomic gene structure (which has one exon and no introns) is shown on the top with start and stop codons bold and underlined. Green sequences are the target sites of guide RNAs while the blue sequence (CCT or CCA) represents the PAM (protospacer adjacent motif). Red arrows indicate the expected sites of cleavage by Cas9. Dashes and red letters indicate the deletion/ insertion of nucleotides, respectively. Deletion mutations ranged from few base pair to more than 900 bp deletion. Numbers on the right side of each sequence indicate the number of nucleotides that have been deleted (−) or inserted (+). Double slash is present where a DNA sequence between two gRNA targets has been omitted for simplicity. When double slash is absent, this means that there is a large deletion and the entire sequence between two gRNA targets has been deleted. Mutations are reported in order starting with those at the 5′ end e.g. + 2-128-39 means 2 bp insertion, 128 bp deletion and 39 bp deletion. Each of the mutant alleles was detected once in 60 sequencing reactions (1/60). 1Aa and 1Ab were cropped and the full-length gels are presented in Supplementary Figure S1A and S1B, respectively.
