Precise CRISPR-Mediated Mecp2 Editing

Two CRISPR RNAs (crRNAs) and ssODNs with 5′- and 3′-homology arms (each 60 mer) flanking loxP variants (lox66 or lox71) were designed to target Mecp2 intron 2 and 3, based on our previous report (Table 1) [10 (link)]. To facilitate the detection of correct insertions, the ssODNs were engineered to contain an NheI restrictionsite and an EcoRI restriction site, respectively, in addition to the loxP sequences. Equal volumes of crRNA (100 μM; IDT, Coralville, IA) and trans-activating crRNA (tracrRNA) (100 μM; IDT) were combined in a duplex buffer (IDT), heated in a thermal cycler to 95 °C for 5 min, and then placed for 10 min at room temperature according to the manufacture’s protocol. Combined crRNA/tracrRNA (3 μM) was mixed with recombinant Cas9 protein (100 ng/μL; GeneArt Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Waltham, MA, USA) and ssODNs (400 ng/μL) in Opti-MEM I (Life Technologies, Carlsbad, CA, USA), and the mixture was placed for 10 min at room temperature before EP.