Abstract
CRISPR/Cas9 mediated genome-editing (GE) technology is a very powerful tool for the easy and rapid development of genetically modified animals. Although the off-target effects of GE were primarily of concern, diverse and frequent unintentional mutations were found in the so-called “on-target” site in generating knock-in mice with a single nucleotide substitution at the H1 enhancer Hox binding site (HBS) of Myf5 locus. Here, we report on the transition of these mutations to the next generation.
In zygotic GE, ribonucleoprotein complexes of Cas9 nuclease and 2 guide RNAs flanking HBS, alongside with a donor single-stranded DNA were electroporated at one-cell. After the 2-cell eggs were transplanted into the oviduct of pseudo pregnant mice, 15 offspring (N0) was obtained. Furthermore, N1 was obtained by mating N0 with the wild type. The target region was amplified by PCR using the tail DNA as a template, and TA cloning was performed to analyze the sequence.
The number of mutant allele types detected in most N1 mice was lower than that in N0 mice corresponding to each N1 mouse. However, not only the knock-in type but also unintentional mutations such as large deletion type and tandem knock-in type were detected in N1. Therefore, under the conditions of this study, most of the various on-target mutations were transmitted to the next generation without exclusion. These potential features of GE technology strongly suggest that proper selection for the purpose is essential for ensuring the safety of GE foods and medicines.
