CRISPRoff is an epigenome-editing system that establishes stable transcriptional repression through targeted DNA methylation without altering the underlying genomic DNA sequence (Nuñez et al., 2021). Because the efficiency of CRISPRoff-mediated repression depends on guide RNA selection, experimentally validated guide RNAs are required for efficient silencing of individual target genes. The Akt family consists of three closely related serine/threonine kinases, AKT1 (PKBα), AKT2 (PKBβ), and AKT3 (PKBγ), which share many functions in regulating cell survival, proliferation, and metabolism while also exhibiting distinct isoform-specific roles (Manning and Toker, 2017). Although several pharmacological Akt inhibitors are available, they generally inhibit multiple Akt isoforms rather than AKT1 specifically (Heerding et al., 2008). In addition, unlike transient siRNA-mediated knockdown, CRISPRoff has the potential to establish stable and long-term gene repression through DNA methylation. Therefore, efficient guide RNAs for CRISPRoff-mediated silencing of AKT1 would provide a useful tool for studies of Akt signaling. However, guide RNAs optimized for CRISPRoff-mediated repression of the human AKT1 gene have not been reported.

The major AKT1 promoter (p1@AKT1) was identified based on the FANTOM5 promoter atlas, in which transcription start sites and promoter activity were mapped using cap analysis of gene expression (CAGE) (FANTOM Consortium and the RIKEN PMI and CLST, 2014). The major FANTOM5 promoter and exon 1 of AKT1 are located within a CpG island, a genomic feature frequently associated with gene promoters (Deaton and Bird, 2011). The CpG island region was defined according to the CpG Islands annotation in the UCSC Genome Browser, which identifies CpG islands based on sequence length, GC content, and the observed-to-expected CpG ratio (Gardiner-Garden and Frommer, 1987). We therefore focused our guide RNA design on the promoter region surrounding the major FANTOM5 promoter. Candidate guide RNAs were identified using the IDT CRISPR-Cas9 guide RNA design tool based on the genomic sequence surrounding the major FANTOM5 promoter. Ten guide RNAs were selected considering their predicted on-target activity, off-target potential, and distribution across the promoter region. Their silencing activities were evaluated by co-transfecting HEK293 cells with CRISPRoff-v2.1 and individual guide RNA expression plasmids, followed by puromycin selection, expansion, and immunoblot analysis of endogenous Akt1 protein expression. Among the ten guide RNAs tested, g1 produced the strongest reduction in Akt1 protein levels. Guide RNAs g4, g5, g7, and g9 also reproducibly reduced Akt1 expression, whereas the remaining guide RNAs showed weaker or more variable effects.

Interestingly, guide RNAs targeting closely spaced or partially overlapping genomic sites exhibited markedly different silencing efficiencies. For example, g1, g2, and g3 target overlapping sequences within the AKT1 promoter, yet g1 produced the strongest repression whereas g2 and g3 showed weaker or more variable effects. Previous studies have shown that CRISPRoff activity is influenced by the position of the guide RNA target site relative to the transcription start site (Nuñez et al., 2021). However, the marked differences observed among closely spaced or partially overlapping guide RNAs in the present study suggest that target position alone does not fully determine CRISPRoff silencing efficiency. Guide RNA activity in CRISPR-Cas9 systems is also known to depend on sequence-related features (Moreno-Mateos et al., 2015), suggesting that sequence-dependent differences in Cas9–guide RNA targeting efficiency may contribute to the variation observed here. Consequently, experimental evaluation of multiple candidate guide RNAs remains important, even when only a limited promoter region is available for guide RNA design.

Together, these results establish a CRISPRoff-based tool for stable repression of human AKT1. The validated guide RNAs reported here provide a useful resource for future studies of Akt signaling and should facilitate the development of CRISPRoff-based experimental systems for investigating isoform-specific functions of AKT1.

Cell culture and transfection HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells were co-transfected with CRISPRoff-v2.1 and the indicated guide RNA expression plasmids using polyethylenimine MAX (PEI MAX; Polysciences). Twenty-four hours after transfection, puromycin (4 μg/mL) was added to the culture medium. Cells were selected for 5–7 days until all non-transfected control cells had been eliminated and were then expanded for an additional 1–2 weeks before analysis. Immunoblotting Cells were lysed directly in SDS sample buffer. Protein concentrations were determined using an SDS-compatible Bradford protein assay. Equal amounts (20 μg) of total protein were subjected to SDS-PAGE and immunoblotting using anti-Akt1 (C73H10, #2938; Cell Signaling Technology) and anti-GAPDH (#60004-1-Ig; Proteintech) antibodies. After incubation with HRP-conjugated secondary antibodies, immunoreactive bands were visualized using Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan) and detected with a Luminograph III imaging system (ATTO, Tokyo, Japan). Band intensities were quantified using ImageJ (NIH). Quantitative data represent three independent experiments.

Plasmids

CRISPRoff-v2.1 (Addgene #167981) Ten guide RNA expression plasmids (pSG-puro-g1–g10) targeting the human AKT1 promoter were generated in this study. Guide RNA sequences are provided in Table 1.

Cell line

HEK293 (ATCC CRL-1573)