CRISPR/Cas9 nickase‐mediated efficient and seamless knock‐in of lethal genes in the medaka fish Oryzias latipes

Abstract

Genome editing with CRISPR/Cas9 system has become a powerful technology for targeted modification of chromosomal sequences in a wide range of organisms. This system consisting of Cas9 and guide RNA (gRNA) can introduce the double strand DNA breaks (DSBs) into the genomic target sites, consequently activating the DNA repair pathways such as non-homologous end-joining (NHEJ) and homology-directed repair (HDR) (Goodarzi & Jeggo, 2013; Sander & Joung, 2014). Although HDR is more ideal than NHEJ for the seamless and specific integration of foreign genes into genomes, generally HDR is not the dominant repair pathway (Han & Huang, 2020; Lieber et al., 2003; Mao et al., 2008). Thus, a majority of DSBs are repaired by error-prone NHEJ rather than the accurate HDR, and this could result in complex and unpredicted genetic mutations, including small insertions and deletions (indels) or imprecise insertion of a donor DNA, into the genomes (

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

“…Thus, this is the first report demonstrating that L755507 promotes HDR events in vivo. In contrast, we did not observe a significant increase in the that, unlike DNA DSB-dependent strategies using wild-type Cas9, the nicking-based approach via Cas9n prevented the activation of NHEJ in medaka as well as other organisms or cells (Gao et al, 2017;Murakami et al, 2020;Paulsen et al, 2017;Y. Wang et al, 2018).…”

“…A group kept at 28 C throughout the experiment was used as the control. In the experiment targeting pdia6 (Figure 1c), we observed green fluorescence in the embryos at 5 days post-fertilization (dpf) and assayed the knock-in efficiencies following the same standard as in our previous study (Murakami et al, 2020). Briefly, the embryos were categorized into the following three groups: "No GFP," "Weak," or "Strong," which represent no fluorescence, less than 40% fluorescence, or more than 40% fluorescence of the body of the embryo, respectively (Figure 1d).…”

“…In our previous study, we demonstrated that the Cas9 nickase (Cas9n)-based strategy enabled the HDR-mediated knock-in of lethal genes by reducing excess gene disruptions at on-and/or off-target sites in medaka (Murakami, Futamata, Horibe, Ueda, & Kinoshita, 2020). While this approach permits expansion of the editable regions including those in lethal genes, the techniques for HDR enhancement were mostly evaluated via combination with knock-in methods using Cas9 and not Cas9n.…”

“…To assess the effects of cold or chemical treatment, we selected pdia6 and gap43 as the target genes and integrated green or red fluorescent protein (GFP or RFP) gene into loci. This experimental procedure allowed us to confirm the successful knock-in of the reporter genes by simply observing the fluorescence of the embryonic bodies under a microscope (Murakami et al, 2017(Murakami et al, , 2020. Finally, employing the optimized conditions, we tested the simultaneous integration of GFP and RFP genes at the pdia6 and gap43 loci, respectively, that is, the double knock-in.…”

A modified Tet‐ON system minimizing leaky expression for cell‐type specific gene induction in medaka fish

“…In addition, 3-dimensional computer graphic animations (Nakayasu et al, 2017) based on the morphologic features and motion characteristics of real fish have been used for studies of medaka social behavior (Shimmura et al, 2017). More recently, several efficient approaches for targeted gene knock-in using the CRISPR/Cas9 system was established in medaka fish (Murakami et al, 2017(Murakami et al, , 2020Watakabe et al, 2018), providing more simple and efficient technology to mimic the expression of endogenous target genes in transgenic strains. The combination of the Tet-ON system and the knock-in system will allow for temporally-controlled induction of a reporter gene under a targeted endogenous promoter, which will be a powerful tool for investigating the neural circuits of social recognition and social behavioral choices using medaka fish.…”