Xenbase Image ID: 157697

Fig. 1. Assessment of CRISPR/Cas9 targeting efficiency of Myod1 by genotyping. Embryos at the one-cell stage were co-injected with 1 ng Cas9 protein and 300 pg of MyoD gRNA. At NF stage 25 genomic DNA was extracted and a 432 bp region including the predicted CRISPR target site was amplified by PCR and cloned into pGEM T-Easy. 3–15 clones per embryo were sequenced and a total of 153 sequences were analysed. (A) The proportions of mutated vs wild type sequences identified in individual embryos is shown as a bar graph. 88.6% of injected embryos have at least one clone mutated at the target sequence. (B) The overall proportion of mutated sequences identified in targeted embryos. An average of 78% of clones per embryo were mutated. (C) Characterisation of mutation types shows the frequencies of insertions/missense as compared to deletions. (D) 10 sequences from a single Cas9 targeted embryo indicate the level of mosaicism present in F0 individuals. Sequences were aligned to the predicted wild type amplicon sequence (bottom row) and the Cas9 PAM sequence is indicated in red underline. (E) Individual sequences were assessed for deletions, insertions and missense mutations. Mutations causing INDELs in multiples of 3 were categorised as in-frame deletions/insertions. 25.9% sequences returned were confirmed wild type, 35.1% showed frame shift deletions, 8.6% showed frame shift insertions, 24.1% showed in frame deletions, 2.3% showed in frame insertions and 4% showed missense mutations. (Blue indicates mutation or disruption; orange indicates wildtype sequence or silent mutation). Image published in: McQueen C and Pownall ME (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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