In vivo evaluation of guide-free Cas9 induced genomic damage and transcriptome changes using pig models (RNA-seq)

The CRISPR/Cas9 system is currently the most promising tool for treating human genetic diseases, several of which have been evaluated in clinical trials with anticipated beneficial effects. Significant potential safety concerns induced by the CRISPR/Cas9 system seriously hinder the clinical trials of gene therapy. Recent reports showed that guide-free Cas9 could induce the genomic instability, activate the p53 pathway in cell levels in vitro. However, the guide-free Cas9-medaited safety concerns have not been evaluated in vivo, which is a necessity for in vivo gene therapy. Here, by using a doxycycline inducible Cas9 expressing pig line, we found that short-term expression of Cas9 alone could induce genomic damage and activate DNA damage repair response in vivo, especially in cells/tissues with high Cas9 expression, such as peripheral blood cells, stomachs and intestines. In these cells/tissues, cellular senescence, apoptosis, immune response, and DNA damage repair response related signal pathways were also activated. The severity of genomic damages and transcriptome changes was correlated with the Cas9 expression levels. These in vivo data of guide-free Cas9 in pigs suggested that as genome editing with the CRISPR/Cas9 system is implemented in the clinic practice of gene therapy, the detrimental effects caused by Cas9 alone should be considered and monitored.