153 Cas9-expressing cattle using all-in-one CRISPR/Cas9 for bovine genome editing

Livestock, particularly cattle, have been central to various biotechnology fields owing to their role in human protein production. However, progress in genetic engineering has been slow, largely because cattle have long gestation periods, typically produce one embryo per pregnancy, and are costly to raise. Thus, we report the birth of transgenic cattle expressing Cas9 and suggest that their derived resources (somatic and germ cells) could be widely used for genome editing in cattle. For the production of Cas9-expressing cattle (F0), a total of 794 oocytes were microinjected with the PB-Cas9-RFP-FatI vector, resulting in 151 blastocysts, of which 34 expressed RFP. From these, four calves (R1, R2, R3, R4) were born. In another experiment, 424 zygotes were microinjected with the PB-Cas9-GFP-sgPRNP vector, producing 84 blastocysts, 31 of which expressed GFP. This resulted in five normal births (G2, G3, G4, G5, G7) and two stillbirths (G1, G6). Genomic PCR confirmed Cas9 integration in all PB-Cas9-RFP-FatI cattle, while PB-Cas9-GFP-sgPRNP integration was found in calves G1, G3, G4, G6, and G7. Fluorescence-positive cell ratios were measured as follows: R1, 58.3%; R2, 33.7%; R3, 87.0%; R4, 74.8%; G1, 87.8%; G2, 0.3%; G3, 42.0%; G4, 16.0%; G5, 0.1%; G6, 0.1%; and G7, 15.4%. Deep sequencing revealed PRNP mutation ratios in PB-Cas9-GFP-sgPRNP cattle as follows: G1, 4.1%; G2, 0.0%; G3, 48.3%; G4, 0.2%; G5, 0.0%; G6, 99.6%; and G7, 94.4%. No off-target effects were detected at the five PRNP off-target candidate loci when targeting the whole genome of cattle, as determined by adjusting the mismatch number to three using the T7E1 assay. Germline transmission was confirmed in SNU-F0-Cas9-RFP-FatI and SNU-F0-Cas9-GFP-sgPRNP cattle, with RFP and GFP expression observed in offspring, respectively, and successful PRNP mutations passed to the next generation. In the application of somatic and germ cells from Cas9-expreesing cattle, Cas9-expressing somatic cells effectively induced targeted gene mutations and were successfully used in somatic cell nuclear transfer, resulting in normal blastocyst development (20.1 ± 11.0%). Knock-in efficiency at the BSA locus exon 1 was significantly improved to 54.17 ± 11.79%, with the insertion of Attb-AfIII sequences and a blastocyst development rate of 29.67%, compared with previous studies showing ~40% knock-in efficiency and 11% embryo development rate. In conclusion, these data demonstrate that, for the first time, Cas9-expressing cattle were born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, F0- and F1-derived somatic and germ cells were used to assess the possibility of gene editing (knockout and knock-in) in several genes, and PRNP-mutated F1 cattle are currently being raised to become the resistance model of bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will be used as a powerful resource for in vitro and in vivo genome editing for a better genetic understanding of bovine genomics and diseases.