Abstract
Genome-editing (GE) technology by using the CRISPR/Cas9 System is expected to greatly promote the development of genetically modified animal models in toxicology. We applied this technique to develop “extra rib” model mice by the strategy of modifying H1 enhancer Hox binding site (HBS) of Myf5, a transcription factor controlling rib formation, into snake type. Although the GE has been concerned for off-target effects, so-called on-target unintentional mutations have not been sufficiently investigated. Here we report the unintentional on-target mutations in detail.
The mouse HBS was edited into snake type by substituting 2 nucleotides. Ribonucleoprotein complexes of Cas9 nuclease and 2 guide RNAs flanking HBS, alongside with a donor single-stranded DNA were introduced into one-cell fertilized eggs by electroporation. The 2-cell eggs were transplanted into the oviduct. The targeted region of GE was TA cloned and more than 10 clones were sequenced for each mouse.
Transplanting 117 embryos resulted 15 offspring mice (N0M). The intended mutation was detected in 6 N0M, while all N0M harbored over 30 types total of unintended mutations, including large deletion and multi-copy insertion of donor DNA. Most N0M did not have wild-type allele and showed mosaic genome including 2-5 types of mutated sequences, suggesting the activity of Cas9 nuclease remained until 8-cell stage. Thus, the on-target unintentional mutations were revealed to be significantly diverse and frequent in GE in our current experiment. Further analysis in the case of the next generation (N1) is now in progress.
