HH5 Double-Carrier Embryos Fail to Progress through Early Conceptus Elongation.

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause pre-term mortality. Holstein Haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the Transcription Factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated to the inadvertent cross between carriers. To determine if HH5 DC survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. DC E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic (>26-fold) reduction in the proliferation of extraembryonic membranes compared with carrier or non-carrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos -functionally equivalent to DC embryos- were generated by CRISPR technology and cultured to the blastocyst stage -Day (D)8- and to the early embryonic disc stage -D12-. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mtDNA content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, required for pregnancy recognition.

RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.

Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single-stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.

Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10-30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.

CRISPR is knocking on barn door.

Genome modification at specific loci in livestock species was only achievable by performing homologous recombination in somatic cells followed by somatic cell nuclear transfer. The difficulty and inefficiency of this method have slowed down the multiple applications of genome modification in farm animals. The discovery of site-specific endonucleases has provided a different and more direct route for targeted mutagenesis, as these enzymes allow the ablation (KO) or insertion (KI) of specific genomic sequences on a single step, directly applied to zygotes. Clustered regularly interspaced short palindromic repeats (CRISPR), the last site-specific endonuclease to be developed, is a RNA-guided endonuclease, easy to engineer and direct to a given target site. This technology has been successfully applied to rabbits, swine, goats, sheep and cattle, situating genome editing in livestock species at an attainable distance, thereby empowering scientist to develop a myriad of applications. Genetically modified livestock animals can be used as biomodels to study human or livestock physiology and disease, as bioreactors to produce complex proteins, or as organ donors for transplantation. Specifically on livestock production, genome editing in farm animals may serve to improve productive genetic traits, to improve various animal products, to confer resistance to diseases or to minimize the environmental impact on farming. In this review, we provide an overview of the current methods for site-specific genome modification in livestock species, discuss potential and already developed applications of genome edition in farm animals and debate about the possibilities for approval of products derived from gene-edited animals for human consumption.