CRISPR-Cas9 Genome Editing of Mecp2 and Tet3 Genes

Two gRNAs targeting Mecp2 intron 2 and intron3 (Table S6) were designed, as well as corresponding lox site ssODNs with 60 bp homology to sequences on each side of each gRNA-mediated DSB (Table S6). To facilitate the detection of correct insertions, the ssODNs targeting intron 2 and the ssODN targeting intron 3 were engineered to contain a NheI restriction site and an EcoRI site, respectively, in addition to the lox sequences. Two gRNAs targeting Tet3 intron 7 and intron 9 (Table S6) and two loxP site ssODNs, containing a BamHI or a EcoRI restriction site with 60 bp homology sequences, were also designed (Table S6). gRNAs were synthesized as previously described^{16 (link)}. Recombinant Cas9 protein (100 ng/μl; GeneArt Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Waltham, MA), gRNA (24 ng/μl), and ssODNs (400 ng/μl) (Table S6) were mixed in RNase-free water for microinjection or in Opti-MEM I (Life Technologies, Carlsbad, CA) for electroporation.