RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Abstract

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 singlestranded 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.

Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

“…After microinjection of the CRISPR components and an ssDNA repair template, in vitro fertilized zygotes were incubated in 7.5 µM RS-1. KI rates were doubled compared to embryos not treated with RS-1 [61]. Another recent study, however, did not observe an effect with RS-1 [41].…”

Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates

“…In this perspective, early TE specification could have emerged as an evolutionary mechanism in rodents to allow implantation at an earlier stage than in other species. Fortunately, recent improvements in genome edition techniques in ungulates ( 39 , 40 ) currently allow the exploration of the molecular machinery involved in ICM/TE specification in farm animals. Genome editing constitutes also an unvaluable tool to study other reproductive processes ( 41 ).…”

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

“…Small molecules treatments to increase KI efficiency have been reviewed (Yeh et al, 2019;Bischoff et al, 2020). No data was reported to date in rats or mice, and only two studies showed that RS-1 enhances KI efficiency in rabbit (Song et al, 2016) and bovine embryos (Lamas-Toranzo et al, 2020). Finally, tests on cells and mouse embryos have shown that ExoI overexpression enhances KI activity (Aida et al, 2016).…”