Guide-free Cas9 is detrimental to pig preimplantation embryo development

Germline genome editing by the CRISPR/Cas9 system has potential for generation of genetically modified animals/the targeted correction of pathological mutations, understanding the mechanism of early embryonic development. Previous reports showed that Cas9 protein without sgRNAs could induce genomic damage in cell levels in vitro. However, whether the detrimental effects occurred in embryos after Cas9-treatment remains unknown. Here, by using pig embryos as the subjective, we found that Cas9 protein transcribed from injected Cas9 mRNA could persist to at least blastocyst stage. Cas9 protein alone could induce the genome damage of preimplantation embryos, represented by increased phosphorylated histone H2AX (pH2AX) foci on the chromatin fiber, leading to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs could also cause the blastocyst transcriptome changes, depressing the embryo development signal pathway, such as cell cycle, metabolism, and cellular communication related signal pathways, while activating apoptosis and necroptosis signal pathway, both of which together resulted in impaired the preimplantation embryonic development.