Ablation ofOCT4function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)

CRISPR-Cas ribonucleoproteins are important tools for gene editing in pre-implantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A ribonucleoproteins targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1000 nucleotides long. Electroporation of Cas13a ribonucleoproteins prevents the production of the corresponding proteins. We electroporated Cas9D10A ribonucleoproteins targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a ribonucleoproteins targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P<0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4 , revealed a significant (FDR<0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency ( CADHD1 , DPPA4 , GNL3 , RRM2 ). The results confirm that OCT4 is key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle. Significance Statement CRISPR-Cas mediated DNA editing can revolutionize agriculture and biomedicine due to its simplicity of design and use. Modifications induced in embryos, though challenging to accomplish, are beneficial for the advancement of livestock production and the study of biological function. Here, we developed an approach using CRISPR-Cas enzymes to remove DNA segments of the cattle genome in one-cell embryos. Our results show major advancement in the efficiency of producing large deletions in the genome of cattle embryos. Using our approach, we removed the function of the OCT4 gene. Our results confirmed OCT4 as a major regulator of pluripotency genes during embryo development and its requirement for the formation of an inner cell mass in cattle.