CRISPR-Cas9 Mediated Genome Editing

Pronuclear injection with 5 ng/μL of gRNA/Cas9 expressing plasmid (for Pate1, Pate2 and Pate3) was performed as previously reported (Mashiko et al., 2013 (link); Noda et al., 2017 (link)). The crRNA and tracrRNA (Sigma) were diluted with nuclease free water (non-DEPC treated, Ambion). The mixture was denatured at 95°C for 1 minute and allowed to anneal by cooling gradually to room temperature (~1 hour). Each gRNA was mixed with Cas9 protein solution (Thermo Fisher Scientific) and T₁₀E_0.1 buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4), and then incubated at 37°C for 5 minutes to prepare the gRNA/Cas9 RNPs. For multiple gene targeting, we prepared the gRNA/Cas9 RNP solution separately and then combined them [final concentration: 30 ng/μL (≈ 200 nM) Cas9 for 20 ng/μL (≈ 600 nM) of each gRNA (Table S2)]. After centrifugation at 20,000 g at 4°C for 10 minutes, the mixture was used for pronuclear injection (for Clpsl2, Epp13, Rnase13, Gm1110, Glb1l2, Glb1l3).