An engineered, compact Cas12m variant for multiplexed gene activation in vivo

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an-engineered,-compact-cas12m-variant-for-multiplexed-gene-activation-in-vivo

Data availability

All 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 availability

No custom code was generated for this study.

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Acknowledgements

We thank J. Li (Shanghai Institutes for Biological Sciences, CAS) for kindly providing the OG2 mice. We thank H. Jiang for constructive advice and support during the course of this work.

Funding

This 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 information

Author notes

  1. These authors contributed equally: Hainan Zhang, Lin Yang, Yuling Liu, Dingyi Han, Yingnan Li, Jiajia Lin.

Authors and Affiliations

  1. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Hainan Zhang, Yaru Zhang, Jinhui Li, Tong Li, Na Zhong, Yingsi Zhou & Guoling Li

  2. Institute 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, China

    Lin Yang, Dingyi Han & Yingnan Li

  3. Department of Neurology, Fujian Institute of Neurology, the First Affiliated Hospital, Fujian Medical University, Fuzhou, China

    Lin Yang & Jiajia Lin

  4. Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China

    Yuling Liu, Ganggang Zhang, Xingtao Zhou & Jinhai Huang

  5. NHC Key Laboratory of Myopia and Related Eye Diseases, Key Laboratory of Myopia and Related Eye Diseases, Chinese Academy of Medical Sciences, Shanghai, China

    Yuling Liu, Ganggang Zhang, Xingtao Zhou & Jinhai Huang

  6. Shanghai Research Center of Ophthalmology and Optometry, Shanghai, China

    Yuling Liu, Ganggang Zhang, Xingtao Zhou & Jinhai Huang

  7. Institute of Neuroscience and Guangxi Key Laboratory of Brain Science, Department of Human Anatomy, School of Basic Medical Sciences, Guangxi Medical University, Nanning, China

    Bingyan Wang & Guohe Tan

  8. Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, China

    Jiaxu Hu, Yi Zhang, Shuping Wei, Simiao Liu, Hui Yang & Guoling Li

  9. Guangzhou University of Chinese Medicine, Guangzhou, China

    Jiaxu Hu

  10. HUIDAGENE Therapeutics, Inc., Shanghai, China

    Linyu Shi

  11. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Hui Yang

Authors

  1. Hainan Zhang
  2. Lin Yang
  3. Yuling Liu
  4. Dingyi Han
  5. Yingnan Li
  6. Jiajia Lin
  7. Bingyan Wang
  8. Jiaxu Hu
  9. Yaru Zhang
  10. Yi Zhang
  11. Jinhui Li
  12. Shuping Wei
  13. Simiao Liu
  14. Tong Li
  15. Na Zhong
  16. Ganggang Zhang
  17. Xingtao Zhou
  18. Linyu Shi
  19. Jinhai Huang
  20. Hui Yang
  21. Guohe Tan
  22. Yingsi Zhou
  23. Guoling Li

Contributions

H.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 authors

Correspondence to Hainan Zhang, Jinhai Huang, Hui Yang, Guohe Tan, Yingsi Zhou or Guoling Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Biotechnology thanks Nozomu Yachie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended 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 data

Extended 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 data

Extended 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 < 0.01.

Source data

Extended Data Fig. 4 PAM specificity and transcriptome-wide profiling of denCas12m.

a, Schematic of dCas12m-miniVPR transcriptional activation across all 16 5’-NTTN PAMs recognition using 1xTS-driven mCherry reporter. b, Heatmap quantifying activation breadth across 16 PAMs for the indicated effectors using the crCMV. c-d, Comparative endogenous activation across the HBG (c), and IFNG (d) loci. RT-qPCR quantification shows mRNA fold-enrichment relative to non-targeting (NT) controls following activation by the indicated dCas12 variants across tiled NTTN PAM sites. Top panels illustrate gRNAs distribution relative to the transcriptional start site (TSS). gRNAs represent crRNAs for dhyperCas12a, dCas12m, and denCas12m, or sgRNAs for dCasMINI). e-g, RNA-seq analysis of HEK293T cells treated with the denCas12m-miniVPR (e), dhyperCas12a-miniVPR (f) and dCasMINI (g) programmed with single- or dual-crRNA array targeting the HBB, HBG, and IFNG promoters. h, Number of DEGs identified in e–g for each system and crRNA configuration. DEG means differentially expressed gene. mRNAs identified as significantly differentially expressed (|log2FC | > 1 and FDR or adjusted P value < 0.05) are shown as pink dots, with specifically targeted loci identified by labeled dots. Data represent mean ± s.d. (n = 3 biologically independent experiments).

Source data

Extended Data Fig. 5 Screening of crRNAs targeting the promoter regions of Utrn, Scn1a and reprogramming factor genes.

a, Positions of the crRNAs screened for each target gene. For Utrn and Scn1a, crRNAs are mapped onto the gene structures showing the distal and proximal promoters, transcription start sites (TSS), first exons and the ATG; coordinates are given relative to each TSS. For Oct4, Sox2, Klf4, Myc and Lin28a, crRNA positions are shown relative to a shared coordinate scale upstream of the TSS (bottom). Each pentagon denotes one crRNA, numbered as in b–h, with the orientation indicating the targeted strand. b–h, Activation of endogenous Utrn (b), Scn1a (c), Oct4 (d), Sox2 (e), Klf4 (f), Lin28a (g) and c-Myc (h) by denCas12m-enminiVPR with each individual crRNA, measured by RT-qPCR and expressed relative to the non-targeting (NT) control. Note the logarithmic y axis in d. Red arrowheads indicate the optimal crRNA of each gene. All experiments were performed in N2a cells. Red arrows indicate lead candidates. Data represent mean ± s.d. (n = 3 biologically independent experiments); individual data points are shown.

Source data

Extended Data Fig. 6 PAM compatibility and rational design of multiplex crRNA arrays.

a, Distribution of PAM-compatible target sites within the selected CRISPRa windows of Utrn, Scn1a, Oct4, Sox2 and Klf4. Vertical ticks denote sites compatible with denCas12m (NTTN PAM), dhyperCas12a, dCasMINI and dSpCas9 (NGG PAM), each fused to enminiVPR. Coordinates are relative to the TSS; the enhancer and proximal promoter regions analysed are indicated. b, Number of PAM-compatible guides available to each system within the indicated CRISPRa windows. Shading within each bar denotes the contribution of each window. Among Cas12-based systems, denCas12m provides substantially more targetable sites than dhyperCas12a and dCasMINI at all five loci. c, Rationale for multiplex crRNA array design. Candidate guides tiling the target locus (step 1) were screened individually and those exceeding the activity threshold were retained (step 2). Retained guides were then filtered pairwise for genomic position and spacing (step 3), excluding pairs that were too far apart, redundant in order, or otherwise suboptimal. Final crRNA arrays were assembled from high-activity, well-spaced guide pairs spanning the proximal and distal promoter regions (step 4). d, Activation of Utrn, Scn1a, Oct4, Sox2 and Klf4 by guide combinations delivered with denCas12m-, dhyperCas12a-, dCasMINI- or dSpCas9-enminiVPR. Each point represents the mean of n = 3 biologically independent experiments for one guide combination. Horizontal lines denote the median. The best-performing combinations for denCas12m and dhyperCas12a are annotated. Note the logarithmic y axis (right panel).

Source data

Extended Data Fig. 7 smCRISPRa-mediated efficient utrophin expression via optimized crRNA arrays in humanized DMD mice.

a, Schematics of the multiplex crRNA arrays tested for Utrn. Each array comprises a single U6-driven transcript containing one or more direct repeats (grey diamonds) and spacers (blue boxes, numbered as the individual guides). Arrays include single guides (g1, g5, g14), two-guide and three-guide combinations, and permutations differing only in guide order. A1 (g1-g5) and A2 (g14-g1-g5) denote the arrays selected for subsequent experiments. b, Activation of endogenous Utrn by smCRISPRa delivered with each crRNA array shown in a, measured by RT-qPCR and expressed relative to the non-targeting (NT) control (dashed line). Red arrowheads indicate the selected arrays (A1 and A2). c, Schematic of the single-AAV smCRISPRa-U1/U2 cassette and in vivo experimental procedure in WT or DMD mice via IM delivery. d, Schematics of two humanized DMD mouse models, highlighting mouse (blue) and human (red) exons. e, RT-qPCR analysis of Utrn activation in the TA muscle of both DMD models following smCRISPRa-U1 or -U2 delivery (n = 3 mice). f, Representative immunofluorescence of utrophin (magenta) and laminin (green). Scale bar, 100 μm. g,i, Western blot analysis of utrophin expression from TA muscle lysates in both DMDΔmE50E51, hE50KI (g) and DMDΔmE23, hE23KI (i) cohorts. Dystrophin as mouse control and vinculin as loading control. n = 3 per group. h,j, Quantification of utrophin normalized to vinculin in g (h) and i (j). Data represent mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001. ns, not significant. Panel c created in BioRender.com (L., J. (2026) https://BioRender.com/mj5s00s).

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Extended Data Fig. 8 Transcriptome-wide profiling of smCRISPRa-U2-mediated Utrn activation in humanized DMD mice.

a, Pairwise comparisons of transcriptome profiles from tibialis anterior muscle. Each panel plots log2(TPM) values between two groups; genes significantly up- or down-regulated are shown in red and blue, respectively, with counts indicated. Utrn is labelled in each panel. Groups: wild-type (WT) and DMDΔmE50E51, hE50KI mice treated with saline, with smCRISPRa carrying a non-targeting crRNA (smCRISPRa-NT), or with smCRISPRa carrying the Utrn-targeting array (smCRISPRa-U2). Differential expression was assessed using DESeq2 with thresholds of adjusted P < 0.05 and |log2FC | > 1; n = 4 mice per group. b, RT-qPCR validation of the top 10 computationally predicted off-target sites (OT1-OT10) in TA muscle from mice treated with smCRISPRa-U2 or control, in WT (left) and DMD (right) backgrounds. Expression is normalized to the corresponding saline control. Data are mean ± s.e.m.; n = 4 mice per group. P values were determined using an unpaired, two-tailed Student’s t-test. *P < 0.05; ***P < 0.001; ns, not significant.

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Extended Data Fig. 9 Multiplex gene activation in HEK293T and primary human T cells using smCRISPRa.

a, Schematic of crRNA positions targeting three genes, HBE1, CARD9 and MYOD1. Candidate crRNAs were designed within the -300 bp to +50 bp window relative to the annotated TSS. Colored labels indicate crRNAs targeting HBE1, CARD9 and MYOD1. b, Single-crRNA activation screening of HBE1, CARD9 and MYOD1 in HEK293T cells using denCas12m-enminiVPR and dhyperCas12a-enminiVPR. Five crRNAs were tested for each gene and compared with the non-targeting control. c, Activation of MYOD1 by selected crRNA combinations identified from the single-crRNA screen in b. crRNA combinations were tested in HEK293T cells. d, Four array architectures, crA1-crA4, were generated for denCas12m and dhyperCas12a to simultaneously target HBE1, CARD9 and MYOD1. Colored boxes indicate gene-specific spacers: HBE1 in yellow, CARD9 in cyan and MYOD1 in pink. Numbers indicate crRNA IDs. Diamonds indicate direct repeat sequences corresponding to denCas12m or dhyperCas12a. e, Three-gene activation in HEK293T cells by the multiplex crRNA arrays shown in d. crA1-crA4 were compared between the denCas12m and dhyperCas12a CRISPRa systems. f, In primary human T cells, three-gene activation using selected multiplex crRNA arrays, crA1 and crA2. Activation mediated by denCas12m-enminiVPR was compared with dhyperCas12a-enminiVPR. Data are shown as mean ± s.d., n = 3 biologically independent experiments. P values were determined using an unpaired two-tailed Student’s t-test. ****P < 0.0001; **P < 0.01; *P < 0.05; ns, not significant.

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Extended Data Fig. 10 The denCas12m platform supports both transcriptional activation with alternative activation domains and CRISPRi-mediated repression.

a, Schematics of the activator constructs. denCas12m was fused to either enminiVPR or the compact activation domain eN3x9; dhyperCas12a and dCasMINI were fused to eN3x9. b, Activation of endogenous HBG by each construct with six crRNAs spanning distinct NTTN PAMs (indicated below). c, Schematics of the CRISPRi constructs, in which denCas12m, dhyperCas12a and dCasMINI were each fused to the KRAB repressor domain. d, Repression of BLM, CDK4, CHK1, CHK2 and HER2 in HEK293T cells by each dCas-KRAB construct with single crRNAs/ sgRNAs. e, Repression of multiple genes by multiplex crRNA arrays cr(BLM-CDK4-CHK1) (top) and cr(CHK1-BLM-CHK2) (bottom), delivered as a single U6-driven array with the cognate direct repeats of each system. f, Repression of PCSK9 in Huh-7 cells by individual crRNAs. crRNA positions relative to the PCSK9 TSS are shown above. g, Repression of PCSK9 in Huh-7 cells by single crRNAs (4 and 6) and the two-guide array cr (4-6). Relative RNA levels were measured by RT-qPCR and normalized to the non-targeting (NT) control of each gene. Data are mean ± s.d. (n = 3 biologically independent experiments); individual data points are shown. P values were determined using an unpaired two tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

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Supplementary information

Reporting Summary (download PDF )

Supplementary Tables 1–8 (download XLSX )

Supplementary Tables 1–8 combined into a single workbook with one tab per table. Supplementary Table 1: Amino acid and DR sequences of Cas12m orthologs. Supplementary Tables 2 and 3: Human codon-optimized expression constructs and crRNA array sequences used in this study. Supplementary Table 4: Spacer and PAM sequences of gRNAs used in this study. Supplementary Table 5: RT–qPCR primers used in this study. Supplementary Table 6: Structure-guided analysis of Cas12m engineering sites in predicted Cas12m–crRNA–dsDNA complexes. Supplementary Table 7: Summary of CRISPRa construct features and activation performance in this study. Supplementary Table 8: In silico predicted candidate non-TSs for the Utrn-targeting crRNAs encoded in smCRISPRa-U2.

Supplementary Data 1 (download XLSX )

Statistical source data for Supplementary Figs. 2 and 5–14 (one clearly named tab per figure).

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Zhang, H., Yang, L., Liu, Y. et al. An engineered, compact Cas12m variant for multiplexed gene activation in vivo. Nat Biotechnol (2026). https://doi.org/10.1038/s41587-026-03352-5

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