For targeting of rs27324996, candidate guide RNAs with a predicted cleavage site near rs27324996 were designed by manual inspection, and the corresponding protospacers were cloned into the pGuide plasmid as described above. Each gRNA plasmid was co-transfected with pCas9_GFP into mouse NIH 3T3 cells using TransIT-2020 Reagent (Mirus Bio) according to the manufacturer’s instructions. Two days post-transfection, GFP-positive cells were isolated by FACS, and genomic DNA was isolated using the DNeasy Blood & Tissue Kit (QIAGEN). The region flanking rs27324996 was PCR amplified (F: 5′-TGGGAATGGCTTCTTAGGGC-3′ and R: 5′-CATCCCCAAGCAACTCAACC-3′) using AccuPrime Taq DNA Polymerase (Thermo Fisher Scientific) with the following cycling conditions: 94°C for 2 min, [94°C for 30 sec, 55°C for 30 sec, 68°C for 30 sec] × 40 cycles, 68°C for 5 min. PCR products were purified using the DNA Clean & Concentrator kit (Zymo Research) and analyzed for the presence of indels using the Surveyor Mutation Detection Kit (Integrated DNA Technologies) according to the manufacturer’s instructions. CEL I nuclease-treated PCR products were resolved on a 1.5% agarose gel to detect mutagenesis activity. The gRNA sequence exhibiting the highest
