An effective double gene knock‐in strategy using small‐molecule L755507 in the medaka fish (Oryzias latipes)

Abstract

Homology-directed repair (HDR)-mediated genome editing has become a powerful method for altering chromosomal sequences in a seamless and accurate manner.However, the low efficiency of HDR in most cells hinders the establishment of desired strains harboring accurately modified genomes. To enhance HDR-mediated knock-in events, we explored two approaches, namely low-temperature incubation and chemical compound administration using medaka embryos after microinjection.We validated the performance of each method by calculating the knock-in efficiencies according to the expression area of fluorescent protein in the embryos. The in vivo assay indicated that the reduction in temperature did not promote HDR events, whereas among the nine compounds screened, the small molecule L755507 could enhance the HDR-mediated targeted integration of reporter cassettes. Addi-

Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency

“…2 B). Recently, the DNA-PK inhibitor NU7441, the RAD51-activator RS-1 and the β3-adrenergic receptor agonist L755507 were shown to increase HDR in zebrafish, medaka or other systems ( Aksoy et al, 2019 ; Murakami and Kobayashi, 2022 ). We tested adding these HDR modulators, the proprietary Alt-R™ IDT HDR Enhancer V2 from Integrated DNA Technologies or a DMSO-only vehicle control to the PCR tagging injection mix.…”

“…These short arms can be included in the oligonucleotide primers and incorporated in the template during PCR. Furthermore, recent studies have sought to tip the NHEJ-HDR balance following Cas9 cleavage to favour HDR via small-molecule HDR activators or NHEJ repressors, which appear to increase HDR in zebrafish ( Aksoy et al, 2019 ; Murakami and Kobayashi, 2022 ). We therefore sought to incorporate HDR enhancers in the workflow, by adding them directly to the solution injected into fertilized eggs.…”