In view of the controversy related to the generation of off-target mutations by gene editing approaches, we tested the specificity of TALENs by disrupting a multi-copy gene family of histone variant, H2A.B3, using only one pair of TALENS. This represents the first described knockout of this histone variant.
We are showing that all three mutations are stably inheritable up to the present, 9th generation of KO mice. Most importantly, we have investigated, using exome sequencing, whether TALEN activity induced any off-target mutations in our mouse model. For that, we sequenced the genomes of mice that belong to 3 consecutive generations and compared the results to wild type mice that were used for breeding of our colony. Our results showed that TALEN technology is capable of producing extremely well-targeted mutations in multi-copy genes and did not produce off-target effects that could be identified by exome sequencing. We also showed that wet-lab validation of predicted off-target sites is crucial in order to make valid conclusions about the specificity of targeting. The phenotypic characterization of a H2A.B3 KO and the impact on the fertility and the epigenome of male germ cells that lack H2A.B.3 will also be presented.