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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #386639

Research Project: Development of Gene-editing Technologies in Livestock to Address Agriculturally Relevant Problems

Location: Plant Genetics Research

Title: Effects of the RS-1, RAD51-stimulatory compound 1, and its vehicle DMSO on pig embryo culture

Author
item LUCAS, CAROL - University Of Missouri
item Redel, Bethany
item CHEN, PAULA - University Of Missouri
item SPATE, LEE - University Of Missouri
item WELLS, KEVIN - University Of Missouri
item PRATHER, RANDALL - University Of Missouri

Submitted to: Reproductive Toxicology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/11/2021
Publication Date: N/A
Citation: N/A

Interpretive Summary: Producing pigs with improved agricultural traits or for validating biomedical models for human health by using a DNA knock-in approach in embryos is currently a very inefficient process. Several strategies have been tested to attempt to improve the efficiency of the incorporation of a site-specific DNA sequence into the genome of rabbit and cow embryos, including the use of a specific compound called RS-1. Here, we assessed the effects of RS-1 on pig embryo development and viability. The addition of RS-1 during embryo culture did not have a negative effect on embryo development or the viability of piglets born after embryos were transferred to a recipient sow. The resulting piglets were healthy, did not show any malformations, and almost half of them had edited DNA. Additional research with RS-1 may document improved efficiency in pig DNA knock-in events in the future.

Technical Abstract: Pigs have become an important model for agricultural and biomedical purposes. The advent of genomic engineering tools, such as the CRISPR/Cas9 system has facilitated the production of livestock models with desired modifications. However, precise site-specific modification in pigs through the HDR pathway still a challenge. In mammalian embryos, the use of small molecules to inhibit NHEJ or to improve HDR have been tested, but little is known about their toxicity. The RS-1 compound stimulates the activity of the RAD51 protein, which has a key role in the HDR mechanism, demonstrating to enhance HDR events in rabbits and bovine embryos. Thus, in this study, we evaluated the dosage and temporal effects of RS-1 on porcine embryo development and viability. Additionally, we assessed the effects of its vehicle DMSO during embryo in vitro culture. The transient exposure to 7.5 µM of RS-1 seemed nontoxic to the embryos and did not appear to impact in the viability of the piglets born. Additionally, the low concentrations of its vehicle DMSO did not show any toxicity to in vitro produced embryos. The transient use of RS-1 at 7.5 µM during in vitro culture seems to be the best protocol of choice to reduce the RS-1 effects while attempting to improve HDR in the pig. The direct injection of the CRISPR/cas9 system, combined with strategies to improve the frequency of HDR-mediated targeted modifications, have become an important tool to simplify and accelerate the production of genetically modified livestock models.