The role of BMP4 signaling in trophoblast emergence from pluripotency.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

BACKGROUND: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. RESULTS: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. CONCLUSION: The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes.

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle.

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos.

Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos.

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around 'intentional genomic alterations' and future regulatory actions regarding genome-edited animals.

Author Correction: Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes.