Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART).

CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation 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 RNPs 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 1,000 nucleotides long. Electroporation of Cas13a RNPs prevents the production of the corresponding proteins. We electroporated Cas9D10A RNPs targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a RNPs 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 (False Discovery Rate, 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 a key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.

Sexing of cattle embryos using RNA-sequencing data or polymerase chain reaction based on a complete sequence of cattle chromosome Y.

When necessary, RNA-sequencing data or polymerase chain reaction (PCR) assays can be used to determine the presence of the chromosome Y (ChrY) in samples. This information allows for biological variation due to sexual dimorphism to be studied. A prime example is when researchers conduct RNA-sequencing of single embryos, or conceptuses, prior to the development of gonads. A recent publication of a complete sequence of the ChrY has removed limitations for the development of these procedures in cattle, otherwise imposed by the absence of a ChrY in the reference genome. Using the sequence of the cattle ChrY and transcriptome data, we conducted a systematic search for genes in the ChrY that are exclusively expressed in male tissues. The genes ENSBIXG00000029763, ENSBIXG00000029774, ENSBIXG00000029788, and ENSBIXG00000029892 were consistently expressed across male tissues and lowly expressed or absent in female samples. We observed that the cumulative values of counts per million were 2688-fold greater in males than the equivalent values in female samples. Thus, we deemed these genes suitable for the sexing of samples using RNA-sequencing data. We successfully used this set of genes to infer the sex of 22 cattle blastocysts (8 females and 14 males). Additionally, the completed sequence of the cattle ChrY has segments in the male-specific region that are not repeated. We designed a pair of oligonucleotides that targets one of these non-repeated regions in the male-specific sequence of the ChrY. Using this pair of oligonucleotides, in a multiplexed PCR assay with oligonucleotides that anneal to an autosome chromosome, we accurately identified the sex of cattle blastocysts. We developed efficient procedures for the sexing of samples in cattle using either transcriptome data or their DNA. The procedures using RNA-sequencing will greatly benefit researchers who work with samples limited in cell numbers which are only sufficient to produce transcriptome data. The oligonucleotides used for the accurate sexing of samples using PCR are transferable to other cattle tissue samples.

Cleavage kinetics is a better indicator of embryonic developmental competency than brilliant cresyl blue staining of oocytes.

In vitro production of embryos (IVP) is a valuable technology to produce embryos of high genetic value. Despite advances in IVP, the efficiency of culture systems remains low. One method to increase IVP success is the early selection of oocytes or embryos that may have greater developmental potential. Here, we investigated two methods of selection, namely BCB staining and cleavage kinetics, both individually and in conjunction, for improved developmental outcomes in vitro. We hypothesized that a synergistic use of both BCB staining and cleavage kinetics would result in identification of embryos of greater developmental potential. The selection of oocytes by BCB staining does select for those oocytes with higher developmental potential, as noted by a greater blastocyst development between BCB positive (32.6%) and BCB negative (22.0%) on day 8 post-fertilization. However, the utilization of BCB staining and cleavage kinetics in tandem resulted in a complete masking of the effect observed when using BCB alone. We obtained the highest proportion of blastocyst development per selection group using cleavage kinetics alone, in which 53.1% of embryos grouped as Fast produced a blastocyst, which was significantly different from the three other groups (Fast+, Slow, not cleaved). We observed, however, that the separation of embryos by cleavage kinetics did not predict their survival to cryopreservation. In conclusion, in standard culture systems, cleavage kinetics is an effective method for the selection of embryos with increased developmental potential to develop blastocysts, however, it may not be effective to select healthy embryos for transfer following cryopreservation.

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

BACKGROUND: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence. METHODS: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes. RESULTS: The BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex. CONCLUSIONS: If oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.