Data availabilityAll sequencing data were deposited to the NCBI Sequence Read Archive under BioProjects PRJNA1492244 and PRJNA1524337. All plasmids generated in this study are available from the corresponding authors upon request and some were deposited to Addgene (262479–262495). Source data are provided with this paper.Code availabilityNo custom code was generated for this study.ReferencesMcCutcheon, S. R., Rohm, D., Iglesias, N. & Gersbach, C. A. Epigenome editing technologies for discovery and medicine. Nat. Biotechnol. 42, 1199–1217 (2024).Article PubMed CAS Google Scholar Bendixen, L., Jensen, T. I. & Bak, R. O. CRISPR–Cas-mediated transcriptional modulation: the therapeutic promises of CRISPRa and CRISPRi. Mol. Ther. 31, 1920–1937 (2023).Article PubMed PubMed Central CAS Google Scholar Matharu, N. et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science 363, eaau0629 (2019).Article PubMed CAS Google Scholar Liao, H. 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Jiang for constructive advice and support during the course of this work.FundingThis work was supported by the Noncommunicable Chronic Diseases National Science and Technology Major Project (2024ZD0537800), National Natural Science Foundation of China (32571683, 32525004), Natural Science Foundation of Shanghai (25ZR1402557), Innovative Drug Research and Development National Science and Technology Major Project (2025ZD1802600, 2025ZD1802000), Brain Science and Brain-Like Intelligence Technology National Science and Technology Major Project (2021ZD0200900), Basic Frontier Scientific Research Program of the CAS from 0 to 1 original innovation project (ZDBS-LY-SM001), Grants from Science and Technology Commission of Shanghai Municipality (23HC1401100), Anhui Provincial Science and Technology Innovation Tackling Program (Project No. 202523n10050014), Eye and ENT Hospital of Fudan University High-level Talents Program (2021318), Joint Laboratory Fund of New Technologies in Ophthalmology and Optometry (hx26023) and Program for Professor of Special Appointment (Eastern Scholar, TP2022046).Author informationAuthor notesThese authors contributed equally: Hainan Zhang, Lin Yang, Yuling Liu, Dingyi Han, Yingnan Li, Jiajia Lin.Authors and AffiliationsShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, ChinaHainan Zhang, Yaru Zhang, Jinhui Li, Tong Li, Na Zhong, Yingsi Zhou & Guoling LiInstitute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Chinese Academy of Sciences, Shanghai, ChinaLin Yang, Dingyi Han & Yingnan LiDepartment of Neurology, Fujian Institute of Neurology, the First Affiliated Hospital, Fujian Medical University, Fuzhou, ChinaLin Yang & Jiajia LinEye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, ChinaYuling Liu, Ganggang Zhang, Xingtao Zhou & Jinhai HuangNHC Key Laboratory of Myopia and Related Eye Diseases, Key Laboratory of Myopia and Related Eye Diseases, Chinese Academy of Medical Sciences, Shanghai, ChinaYuling Liu, Ganggang Zhang, Xingtao Zhou & Jinhai HuangShanghai Research Center of Ophthalmology and Optometry, Shanghai, ChinaYuling Liu, Ganggang Zhang, Xingtao Zhou & Jinhai HuangInstitute of Neuroscience and Guangxi Key Laboratory of Brain Science, Department of Human Anatomy, School of Basic Medical Sciences, Guangxi Medical University, Nanning, ChinaBingyan Wang & Guohe TanZhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, ChinaJiaxu Hu, Yi Zhang, Shuping Wei, Simiao Liu, Hui Yang & Guoling LiGuangzhou University of Chinese Medicine, Guangzhou, ChinaJiaxu HuHUIDAGENE Therapeutics, Inc., Shanghai, ChinaLinyu ShiState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, ChinaHui YangAuthorsHainan ZhangView author publicationsSearch author on:PubMed Google ScholarLin YangView author publicationsSearch author on:PubMed Google ScholarYuling LiuView author publicationsSearch author on:PubMed Google ScholarDingyi HanView author publicationsSearch author on:PubMed Google ScholarYingnan LiView author publicationsSearch author on:PubMed Google ScholarJiajia LinView author publicationsSearch author on:PubMed Google ScholarBingyan WangView author publicationsSearch author on:PubMed Google ScholarJiaxu HuView author publicationsSearch author on:PubMed Google ScholarYaru ZhangView author publicationsSearch author on:PubMed Google ScholarYi ZhangView author publicationsSearch author on:PubMed Google ScholarJinhui LiView author publicationsSearch author on:PubMed Google ScholarShuping WeiView author publicationsSearch author on:PubMed Google ScholarSimiao LiuView author publicationsSearch author on:PubMed Google ScholarTong LiView author publicationsSearch author on:PubMed Google ScholarNa ZhongView author publicationsSearch author on:PubMed Google ScholarGanggang ZhangView author publicationsSearch author on:PubMed Google ScholarXingtao ZhouView author publicationsSearch author on:PubMed Google ScholarLinyu ShiView author publicationsSearch author on:PubMed Google ScholarJinhai HuangView author publicationsSearch author on:PubMed Google ScholarHui YangView author publicationsSearch author on:PubMed Google ScholarGuohe TanView author publicationsSearch author on:PubMed Google ScholarYingsi ZhouView author publicationsSearch author on:PubMed Google ScholarGuoling LiView author publicationsSearch author on:PubMed Google ScholarContributionsH.Z., G.L., Y. Zhou, J. Huang and H.Y. jointly conceived of the project and designed the experiments. H.Z., L.Y. and Y. Liu performed RNA and protein engineering and reporter assays. H.Z., L.Y. and Y. Liu conducted the endogenous gene-activation-related experiments and RNA-seq assay. H.Z. and L.Y. executed the predictions and analysis of the structures. G.L., J. Lin, S.W. and S.L. performed the DMD animal-related experiments and in vivo multiplexed gene activation experiments. G.T., D.H. and B.W. performed the Dravet animal-related experiments. Y. Li performed the bioinformatics analysis. Yaru Zhang, J. Hu, Yi Zhang, J. Li, S.W., S.L., T.L., N.Z., G.Z., X.Z. and L.S. assisted with the experiments. G.L., Y. Zhou, H.Y., G.T. and J. Huang supervised the whole project. H.Z., H.Y., G.L., Y. Zhou and J. Huang wrote and revised the paper with data contributed by all authors who participated in the project.Corresponding authorsCorrespondence to Hainan Zhang, Jinhai Huang, Hui Yang, Guohe Tan, Yingsi Zhou or Guoling Li.Ethics declarationsCompeting interestsThe authors declare no competing interests.Peer reviewPeer review informationNature Biotechnology thanks Nozomu Yachie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Iterative engineering and functional validation of dCas12m variants in mammalian cells.a, Schematic diagram of the domains within Cas12m18 protein. b, Validation of pre-crRNA processing activity for dCas12m variants identified in binding screens. The decreased fluorescence intensity was normalized to NT as 1. NT, control plasmid lacking the Cas protein. c, Validation of high-potency dCas12m-v1 relative to dCas12m-WT after first round screening in the dual-fluorescence reporter. d, Validation of improved dCas12m variants relative to v1 after second round screening in two independent fluorescence reporters. e, Validation of improved dCas12m variants relative to v2 after third round screening in a mCherry reporter. f, Validation of variants containing V422R and/or Y425R at endogenous HBB and HBG loci. Relative RNA levels are shown for v2, V422R + v2, Y425R + v2 and V422R-Y425R + v2. NT, non-targeting control. Non-target (NT) denotes a spacer with a random sequence. Data represent mean ± s.d. (n = 3 biologically independent experiments). One‑way ANOVA followed by Tukey’s multiple comparisons test was used for pairwise comparisons among all groups. ns, not significant.Source dataExtended Data Fig. 2 Optimization of direct repeat architecture for dCas12m-based transcriptional activation.a, d, Schematic of direct repeat (DR) truncations, ranging from 36-nt (canonical) to 25-nt (a) and 29-nt (d) variants. b-c, Performance of dCas12m variants in separate reporter assays. Mean EGFP (targeting Tet3) and mCherry (targeting miniCMV) fluorescence intensities in HEK293T cells using (b) dCas12m-WT or (c) dCas12m-v3 programmed with crRNAs carrying the indicated DR variants. e-f, Performance of dCas12m variants in dual-fluorescence reporter. Mean EGFP and mCherry fluorescence intensities measured in a dual-reporter assay using (e) dCas12m-WT or (f) dCas12m-v3. g, Schematic of the crRNA architectures containing single crRNA and crRNA array for enhanced gene activation by dCas12m. h-i, Comparison of transactivation efficiency of diverse crRNA scaffold and truncated lengths when paired with either dCas12m-WT (h) or dCas12m-v3 (i) in to separate or a dual-fluorescence reporter system. Non-target (NT) denotes a spacer with a random sequence. Data represent mean ± s.d. (n = 3 biologically independent experiments).Source dataExtended Data Fig. 3 Cas12m possesses crRNA self-processing activity but shows no detectable DNA cleavage activity.a, Schematics of the reporter systems. Left: the DR–EGFP processing reporter, in which cleavage of the direct repeat (DR) embedded in the pre-mRNA generates mature RNA and reduces EGFP output. Right: single-strand annealing (SSA)-based reporters for double-strand break (DSB) formation and for nicking activity in the PAMout and PAMin configurations; cleavage at the target site (GTTC-crTET3) reconstitutes functional EGFP through SSA repair. b, crRNA processing activity of wild-type Cas12m and Cas12m-v3, assessed with DR reporters carrying DRs of 36, 35 and 34 nt. Processing is reflected by reduced normalized mean GFP intensity relative to the non-targeting (NT) control. c-d, Nicking activity measured with the PAMout (c) and PAMin (d) SSA reporters, quantified as the percentage of EGFP+ cells among BFP+ (transfected) cells. Constructs tested include wtCas12m and enCas12m, each with an intact RuvC domain or carrying the RuvC-inactivating substitutions D320A, E473A or D320A-E473A. hyperCas12a and hfCas12Max served as positive controls; dhyperCas12a served as a catalytically dead control. e, DSB activity measured with the SSA-based DSB reporter for wtCas12m, enCas12m and hfCas12Max, each with an intact RuvC domain or the E473A or D320A substitution. f, Indel frequencies at four endogenous HBG target sites (HBG-g1 to HBG-g4) determined by targeted amplicon deep sequencing for hfCas12Max, wtCas12m, denCas12m (D320A-E473A), enCas12m, dhyperCas12a and hyperCas12a. NT, non-targeting crRNA control. Note the discontinuous y axis. All experiments were performed in HEK293T cells. Data are mean ± s.d. (n = 3 biologically independent experiments); individual data points are shown. One‑way ANOVA followed by Tukey’s multiple comparisons test was used for pairwise comparisons among all groups. **P 1 and FDR or adjusted P value