XB-ART-53718Mech Dev. August 1, 2017; 146 1-9.
An analysis of MyoD-dependent transcription using CRISPR/Cas9 gene targeting in Xenopus tropicalis embryos.
PubMed ID: 28536000
Article link: Mech Dev.
Genes referenced: actc1 babam1 bmpr1b cdx1 decr2 dnajc24 epn2 flvcr2 foxc1 foxc2 fstl1 gbx2.2 gli2 herpud2 mrps30 msi1 myod1 nkx6-2 pam pbx2 pcdh8.2 pdlim7 pex16 pgk1 pgp pmm2 pygm rbm20 rbm24 rnf157 rnf7 slc13a4 sp5 sp8 tsfm zeb2
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|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).|
|Fig. 4. Hierarchical clustering of Myod target genes. Genes identified as significantly downregulated in targeted samples were further analysed using expression data from (Tan et al., 2013) to create a heatmap. Relative expression is shown as a scale of low (blue) to high (orange). Euclidean distance was used as the metric for hierarchical clustering of complete samples, which resulted in 5 clusters showing distinct expression profiles. (*) indicates genes which have known or predicted roles in myogenic or pre-myogenic cells. Myod and a-actin (actc1) were included in the analysis to highlight relevant clusters.|