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 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.

SUBMITTER: Lamas-Toranzo I 

PROVIDER: S-EPMC7496720 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature