Development and application of CRISPR technology for the study of reproductive processes in mammals

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Fisiología Animal, leída el 13-01-2021Las aplicaciones de la mutagénesis dirigida se han visto obstaculizadas por dificultades técnicas. La baja eficiencia de la recombinación homóloga (HR), el único método disponible para llevar a cabo esta tarea antes del descubrimiento de las endonucleasas específicas de sitio, hace necesario el uso de células intermedias. Este requisito ha restringido en gran medida la mutagénesis dirigida al modelo del ratón de laboratorio, la única especie en la que se disponía de células madre pluripotentes como células intermediarias. El desarrollo de endonucleasas específicas sitios en los últimos años ha facilitado en gran medida la tarea de lograr una mutación en un lugar específico del genoma. Estas tecnologías incluyen la incorporación más notoria y reciente: CRISPR-Cas9. Mediante esta tecnología, pueden lograrse directamente modificaciones en un lugar específico del genoma mediante la transmisión de los componentes de CRISPR a un cigoto, evitando así la necesidad de células intermedias. Este avance facilita enormemente el proceso en ratones y, especialmente, en otras especies en las que, en ausencia de células madre, se requería la transferencia nuclear de células somáticas [en inglés “somatic cell nuclear transfer” (SCNT)], para generar mutagénesis dirigida. Además, la generación de mutantes en un solo paso permite el estudio de la función génica durante el desarrollo embrionario sin necesidad de mantener colonias de animales mutantes...The applications of targeted mutagenesis have been hampered by technical difficulties. The low efficiency of homologous recombination (HR), the only available method before the discovery of site-specific endonucleases, requires the use of intermediary cells. This requirement has largely restricted targeted mutagenesis to the mouse model, the only species where pluripotent stem cells were available as intermediary cells. The development of site-specific endonucleases in recent years has greatly facilitated the task of achieving a mutation at a specific locus of the genome. These technologies include the latest and most notable incorporation: CRISPR-Cas9. By this technology, site-specific genome modifications can be directly achieved by delivering of CRISPR components into a zygote, thereby avoiding the need of intermediary cells. This improvement greatly facilitates the process in mice and, specially, in other species where, in the absence of stem cells, somatic cell nuclear transfer was required to generate site-specific mutagenesis. Besides, the generation of mutants in a single step allows the study of the gene function during embryo development without the need of maintaining colonies of mutant animals...Fac. de VeterinariaTRUEunpu

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization. © 2020, Lamas-Toranzo et al

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment.We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germlayer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stemcells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestockmammals, pluripotent stemcells related to the formative embryonic disc are reliably established using a common and defined signalling environment

Mitochondrial and metabolic adjustments during the final phase of follicular development prior to IVM of bovine oocytes

In vitro maturation (IVM) leads to reduced developmental rates compared to the use of in vivo matured oocytes. This reduction can be attributed to the suboptimal environment experienced during IVM, but the use of incompetent oocytes also plays a significant role. The objective of this study has been to characterize the mitochondrial and metabolic differences between competent and incompetent bovine oocytes selected prior to IVM based on Brilliant Cresyl Blue (BCB) staining. BCB selection allowed to sort two populations of cumulus-oocyte complexes (COCs) exhibiting diverse developmental competence despite showing a similar size and thereby being morphologically undistinguishable otherwise. Nuclear maturation rates were similar in both populations, but cleavage and blastocysts rates were significantly higher in BCB+ compared with BCB-. Mitochondrial distribution was similar between both groups, but mtDNA content experienced a 1.9-fold increase between BCB- and BCB+ oocytes, suggesting that a significant mtDNA synthesis must occur at the last stages of follicular development to achieve full competence prior to IVM. Consistently, transcriptional analysis in cumulus cells revealed an upregulation of the mitochondrial transcription factor TFAM in BCB-. Transcriptional analysis also suggested a decrease in both anaerobic glycolysis and pentose phosphate pathway (PPP) in BCB+ COCs, as the anaerobic glycolysis enzymes GAPDH and LDHA and the positive regulator of G6PD activity SIRT2 were upregulated in BCB- cumulus cells. These results suggest that during the final stages of follicular development a significant mtDNA replication must occur to achieve full oocyte developmental competence, and that this replication may be linked to anaerobic glycolysis and PPP activities

Directions and applications of CRISPR technology in livestock research

The ablation (KO) or targeted insertion (KI) of specific genes or sequences has been essential to test their roles on a particular biological process. Unfortunately, such genome modifications have been largely limited to the mouse model, as the only way to achieve targeted mutagenesis in other mammals required from somatic cell nuclear transfer, a time- and resource-consuming technique. This difficulty has left research in livestock species largely devoided of KO and targeted KI models, crucial tools to uncover the molecular roots of any physiological or pathological process. Luckily, the eruption of site-specific endonucleases, and particularly CRISPR technology, has empowered farm animal scientists to consider projects that could not develop before. In this sense, the availability of genome modification in livestock species is meant to change the way research is performed on many fields, switching from descriptive and correlational approaches to experimental research. In this review we will provide some guidance about how the genome can be edited by CRISPR and the possible strategies to achieve KO or KI, paying special attention to an initially overlooked phenomenon: mosaicism. Mosaicism is produced when the zygote´s genome edition occurs after its DNA has replicated, and is characterized by the presence of more than two alleles in the same individual, an undesirable outcome when attempting direct KO generation. Finally, the possible applications on different fields of livestock research, such as reproduction or infectious diseases are discussed

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.This work has been funded by the projects RYC-2012-10193, AGL2014-58739-R and AGL2017-84908-R from the Spanish Ministry of Economy and Competitiveness (MINECO) and the project StG-757886-ELONGAN from the European Research Council. I.L.T. is funded by a FPI fellowship by MINECO.Peer reviewe

The human cumulus cell transcriptome provides poor predictive value for embryo transfer outcome

9 Pág.Is the transcriptome of cumulus cells a good predictor of the embryo's developmental competence?This work was funded by the projects IND2017/BIO-7748 from the Madrid Region Government, and AGL2017-84908-R and PID2020-117501RB-I00 from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). A.M.-M. was funded by project IND2017/BIO-7748, and I.L.-T. by an FPI fellowship by MINECO.Peer reviewe

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. © 2017 Blackwell Verlag Gmb