Figure 1
CRISPR/Cas9-based generation of Myh11 (Δ1256K) mice. (A) Sequence of exon 28 of murine Myh11 to which both gRNA1 and 2 bind and an Myh11 (Δ1256K) DNA donor targeting site. The PAM and modified PAM sequence site in the Myh11 (Δ1256K) DNA donor fragment are indicated by red and green, respectively. Locations of PCR primers are indicated by arrows. (B) Table summarising the number (no.) of zygotes injected, no. zygotes transferred and no. zygotes developed to full term is shown. The no. of pups showing knock-in is also shown. (C) Sequences around the Myh11 target site in wild-type (WT) and knock-in samples (nos. 16, 20, 22 and 24). Upper panel shows sequences with the deletion of AAG alone and those in which both PAM sequences (red) were changed to modified PAM sequences (green) in the samples numbered 16, 20 and 24. Lower panel shows the sequences showing the deletion of AAG alone and those in which only a PAM sequence recognised by gRNA1 (red) was changed to modified PAM sequences (green) recognised by gRNA1 in the sample numbered 22. (D) Amino acid sequences around the Myh11 target site of WT and knock-in samples (nos. 16, 20, 22 and 24). Note that the sequences of all knock-in pups are the same as those of the WT pups except for the absence of lysine (K) in knock-in pups. Red shows amino acids corresponding to WT PAM sequences. Green shows amino acids corresponding to modified PAM sequences. The no. 22 line with WT PAM sequences recognised by gRNA2 was selected for further analysis because its alleles are close to the WT Myh11 allele. The no. 22 line was backcrossed to B6 background through passing 2 generations. (E) Myh11 (Δ1256K) genotyping of the no. 22 line by direct sequencing of PCR products corresponding to the Myh11 target site. The blue line shows AAG × 4, the red line shows AAG × 3 and the arrow shows the start of error ideograms.
