Validation of CRISPRoff guide RNAs for stable repression of human AKT1

CRISPRoff enables stable gene repression through targeted DNA methylation, but its efficiency depends on guide RNA selection. To identify effective guide RNAs for human AKT1, we evaluated ten guide RNAs targeting the AKT1 promoter region in HEK293 cells. Guide RNA g1 produced the strongest reduction in Akt1 protein expression, whereas g4, g5, g7, and g9 also reproducibly reduced Akt1 levels. Interestingly, closely spaced or partially overlapping guide RNAs exhibited markedly different silencing efficiencies. These validated guide RNAs provide a useful resource for future studies of Akt signaling.