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Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Nature Cell Biology volume 24, pages 590–600 (2022)Cite this article

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Abstract

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR–Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

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Fig. 1: Development of combinatorial dCas12a mutants with superior activity under conditions of low crRNA levels.
Fig. 2: HyperCas12a outperforms WT dCas12 in CRISPR repression and in vivo gene editing.
Fig. 3: HyperdCas12a enables multiplex activation of endogenous genes.
Fig. 4: In vivo gene activation by hyperdCas12a with single crRNA and poly-crRNA arrays.
Fig. 5: In vivo multiplex gene activation by hyperdCas12a compared with dCas12a alternatives.
Fig. 6: Multiplex gene activation by hyperdCas12a induces the migration of retina progenitor cells and altered differentiation in vivo.

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Data availability

Whole-transcriptome sequencing data can be accessed in Gene Expression Omnibus under the accession code GSE166817. Key constructs and plasmids will be available on Addgene (https://www.addgene.org/Stanley_Qi/). All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The gene-level FPKM values were calculated using a custom Python script available at https://github.com/QilabGitHub/FPKMcalculation. The semi-fluorescence intensities of individual cells (microscopy) were quantitated with a semi-automated image analysis pipeline based on MATLAB (version R2019a) available at https://github.com/QilabGitHub/dCas12a-microscopy.

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Acknowledgements

L.Y.G. acknowledges support from a Stanford’s National Eye Institute (NEI) T32 Vision Training Grant (grant no. 5T32EY020485), the Knights Templar Eye Foundation (KTEF) and the VitreoRetinal Surgery Foundation (VRSF). L.S.Q. acknowledges support from the Li Ka Shing Foundation, National Science Foundation CAREER award (award no. 2046650), National Institutes of Health (grant no. 1U01DK127405), Chan Zuckerberg Initiative Neurodegeneration Challenge Network, Stanford Maternal and Child Health Research Institute (MCHRI) through the Uytengsu–Hamilton 22q11 Neuropsychiatry Research Award Program, Stanford–Coulter Translational Research Grant, and California Institute for Regenerative Medicine (CIRM; grant no. DISC2-12669). S.W. is supported by funding from the American Diabetes Association (grant no. 1-16-INI-16), NIH grant no. 1R01EY03258501, NIH grant no. R01NS109990, NIH-NEI grant no. P30-EY026877 and Research to Prevent Blindness, Inc. BioRender.com was used to generate illustrations in Fig. 1b, 1g, 2a, 2c, 2f, 3a, 4a and 6e. The members of the Qi, Wang and Hu laboratories provided valuable discussion and support for this project.

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Author notes

  1. These authors contributed equally: Lucie Y. Guo, Jing Bian.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Lucie Y. Guo, Jing Bian, Hannah R. Kempton, Xiaowei Zhang, Augustine Chemparathy, Baokun Gu, Xueqiu Lin, Draven A. Rane, Xiaoshu Xu, Ryan M. Jamiolkowski & Lei S. Qi

  2. Department of Ophthalmology, Stanford University, Stanford, CA, USA

    Lucie Y. Guo, Alexander E. Davis, Pingting Liu, Yang Hu & Sui Wang

  3. Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA

    Lei S. Qi

  4. Stanford ChEM-H Institute, Stanford University, Stanford, CA, USA

    Lei S. Qi

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Contributions

L.Y.G. and L.S.Q. conceived the idea for this study. L.Y.G., J.B., S.W. and L.S.Q. designed the experiments. L.Y.G., J.B., X.Z., H.R.K., B.G., D.A.R. and R.M.J. performed the ex vivo experiments. L.Y.G., J.B., A.E.D. and P.L. performed the in vivo experiments with guidance from S.W. and Y.H. L.Y.G., J.B., S.W. and L.S.Q. analysed the experimental data. A.C. and X.L. performed the computational analyses of the sequencing data, and X.Z. and R.M.J. analysed the imaging data. X.X. provided reagents. L.Y.G. and L.S.Q. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Lucie Y. Guo, Sui Wang or Lei S. Qi.

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Competing interests

The authors have filed a provisional patent via Stanford University related to the work (US patent no. 63/148,652). L.S.Q. is a founder and scientific advisory board member of Epicrispr Biotechnologies.

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Nature Cell Biology thanks Stephen H. Tsang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Gating strategies for flow cytometry.

a, Standard strategy for gating cells based on forward scatter (FSC) and side scatter (SSA) (left), then further gating for singlets based on FSC-height (FSC-H) and FSC-area (FSC-A) (right). To analyse transfected cells, further gating is applied to the singlet population based on fluorescence intensity. Please note that for Fig. 1c-e, and Extended Data Fig. 2a–j, that gating strategy is included within the figure. b, Gating strategy for experiments with Cas12a-mCherry plasmid and a CAG-crRNA plasmid (without fluorophore), thus mCherry+ cells are used for analysis. c, Gating strategy for Fig. 1g, in which 3 plasmids are co-transfected. d, Gating strategy for some experiments with Cas12a-mCherry plasmid and U6-crRNA (with BFP). e, Mean BFP fluorescence across the mutants tested in Fig. 1c. f, Mean mCherry fluorescence among mutants tested in Fig. 1c. In e-f, each data point represents the mean GFP intensity of an independent experiment, with each bar representing the average of 2 or more independent experiments. g, Schematic of the LbCas12a protein domains and location of four of the most potent point mutants, with alignment across various Cas12 species. The relevant Asp (D) or Glu(E) residues are highlighted in red.

Source data

Extended Data Fig. 2 Optimizing the nuclear localizing signal and comparing to enAsdCas12a.

a. Schematic to test two different nuclear localization signals. Constructs containing either 2×SV40 or 2×Myc NLS fused with WT dCas12a are co-transfected with Tet crRNA in TRE3-GFP HEK293T reporter cells. b, Representative flow cytometry histogram of BFP intensity, showing threshold for BFP + cells, and subset of ‘low BFP + ’ cells (similar to Fig. 1d). c-d, GFP fluorescence in BFP + (c) or ‘low BFP + ’ cells (d). e-g, GFP fluorescence in BFP + (f) or low BFP + cells (g) to compare WT versus hyperdCas12a (hyp) with 2×Myc NLS, as well as BFP and mCherry average fluorescence in each gated BFP group. h-i, Alignment of the structure of LbCas12a versus AsCas12a proteins (h) and alignment of peptide sequences (i) encompassing mutations harboured by enAsCas12a, a reported enhanced variant of Cas12a from Acidaminococcus with the E174R/S542R/K548R mutations5 corresponding to homologous residues (D156R/G532R/K538R) mutations in LbCas12a. j, Comparison of variants containing mutations of homologous residues in LbCas12a in ‘low BFP + ’ cells. Interestingly, D156R combined with G532R and/or K538R did not achieve activation higher than the single D156R mutant, in contrast to results with homologous residues in AsCas12a5. k, Comparison of hyperdCas12a versus enAsdCas12a with a single crRNA driven by U6 promoter in 1:1 versus 1:0.2 ratio of dCas12:crRNA, in TRE3G-GFP HEK293T cells. l, Comparison of hyperdCas12a versus enAsdCas12a with single crRNA driven by CAG promoter flanked by direct repeats (DR) specific to LbCas12a versus AsCas12a. m, Comparison of hyperdCas12a versus enAsdCas12a with dual crRNAs containing crTet on the second position and non-targeting crLacZ on the first position flanked by As or Lb direct repeats (DR). All transfections in this figure were carried out in TRE3G-GFP HEK293T reporter cells. Bar graph in f, g and k-m shows the mean of n ≥ 3 independent experiments; bar graph in j shows the mean of n ≥ 2 independent experiments; each data point represents value of an independent experiment.

Source data

Extended Data Fig. 3 Improved gene editing by hyperCas12a.

a. Nuclease-active WT Cas12a versus hyperCas12a were co-transfected with crGFP into HEK293T cells stably expressing GFP driven by SV40 promoter. b. Representative flow cytometry histogram showing threshold for mCherry+ cells. Analysis of mCherry+ cells are shown in Fig. 2d, while bulk cells (without sorting) were used for indel analysis (panels c-d). c. Indel activity at each nucleotide position, shown as percentage of total reads with a deletion at the position. The PAM is highlighted in pink. d, Indel patterns and corresponding ratios in total reads detected by deep sequencing as analysed by CRISPResso252.

Extended Data Fig. 4 Characterization of off-target effects of hyperdCas12a.

a. RNA-seq FPKMs (fragments per kilobase million fragments mapped) are plotted as transfected versus non-transfected cells. The transfected samples are TRE3G-GFP HEK293T reporter cells co-transfected with WT dCas12a or hyperdCas12a, and with non-targeting crRNA or crRNA targeting TRE3G. b. RNA-seq plots showing FKPM (Fragments Per Kilobase Million) between two biological duplicates for each condition. The calculated Pearson correlation coefficient for each condition is shown on the graph.

Extended Data Fig. 5 Dual antibiotic selection for co-transfected mouse P19 cells.

a, Mouse P19 cells were co-transfected with constructs expressing puromycin resistance (PuroR) and hygromycin resistance (HygroR), then selected with puromycin and hygromycin at 24 hr after transfection. Cells were collected for analysis 72 hr after transfection. b, Histograms showing percentage of BFP + (crRNA) and mCherry + (dCas12a) cells for non-transfected, non-selected, and Puro/Hygro-selected cells. c, Flow cytometry plots. Data in panels b-c are representative plots of n = 3 independent experiments.

Extended Data Fig. 6 Screening dCas12a crRNAs for activating endogenous Sox2.

a, Schematic of dCas12a crRNAs (red) targeting promoter of Sox2, and their relative positions to validated dCas9 sgRNAs31 that are functional (black) for activating Sox2. Arrows indicate sense or antisense binding of crRNAs/sgRNAs to target DNA. The genomic position of the first ‘T’ in PAM (relative to TSS, which is ‘0’) are shown for each crRNA targeting the Sox2 promoter. b, Immunostaining of Sox2 expression from activation by WT dCas12a–miniVPR with various Sox2 single crRNAs, compared to activation by dCas9-miniVPR (using a validated sgRNA, S84)31. Scale bar, 100 µm. c-d, Immunostaining of Sox2 expression and co-localization with BFP and mCherry for a pair of crRNAs (c) and a panel of ‘triplet’ crRNAs (d), demonstrating additive or synergistic effect when multiple crRNAs are used in tandem. Interestingly, addition of a third crRNA targeting a region between the paired crRNAs S1 and S2 decreases the level of activation. Inset (c) shows brightfield image to demonstrate nuclear localization of mCherry (hyperdCas12a) and target (Sox2), since BFP on crRNA plasmid precludes the use of an additional nuclear dye. White scale bar, 100 µm; yellow scale bar (within inset), 50 µm e-f. Automated quantitation of images in panels b-d. In panel e, 350-2000 cells for each condition were quantitated for one screening experiment with multiple fields of view. The exact number of cells for each condition is listed in the Source Data for Extended Data Fig. 6. NT, non-targeting crRNA. In panel f, 70-250 cells for each condition were quantitated for one screening experiment with multiple fields of view. For box-and-whisker plots, the box shows 25-75% (with bar at median, dot at mean), and whiskers encompass 10-90%, with individual data points shown for the lowest and highest 10% of each dataset. The exact number of cells for each condition are listed in the Source Data for Extended Data Fig. 6. NT, non-targeting crRNA.

Source data

Extended Data Fig. 7 Screening dCas12a crRNAs for activating endogenous Klf4.

a, Schematic of dCas12a crRNAs (red) targeting promoter of Klf4 and their relative positions to known dCas9 sgRNAs31 that are functional (black) or non-functional (grey) for activating Klf4. Arrows indicate sense or antisense binding of crRNAs/sgRNAs to the target DNA. The genomic position of the first ‘T’ in PAM (relative to TSS, which is ‘0’) are shown for each crRNA targeting to the Klf4 promoter. b, Immunostaining of Klf4. Inset shows brightfield image to demonstrate nuclear localization of mCherry (hyperdCas12a) and target (Klf4), since BFP on crRNA plasmid precludes the use of an additional nuclear dye. White scale bar, 100 µm; yellow scale bar (within inset), 50 µm c. Automated quantitation of images in panel b, where 200-600 cells for each condition were quantitated for one screening experiment with multiple fields of view. The exact number of cells for each condition is listed in the Source Data for Extended Data Fig. 7. NT, non-targeting crRNA.

Source data

Extended Data Fig. 8 Screening dCas12a crRNAs for activating endogenous Oct4.

a, Schematic of dCas12a crRNAs (red) targeting promoters of Oct4 and their relative positions to known dCas9 sgRNAs31 that are functional (black) or non-functional (grey) for activating Oct4. Arrows indicate sense or antisense binding of crRNAs/sgRNAs to the target DNA. The genomic position of the first ‘T’ in PAM (relative to TSS, which is ‘0’) are shown for each crRNA targeting to the Oct4 promoter. b, Immunostaining of Oct4. White scale bar, 100 µm. c, Inset shows merge with brightfield to demonstrate nuclear localization of mCherry (hyperdCas12a) and target (Oct4), since BFP on crRNA plasmid precludes the use of an additional nuclear dye. Yellow scale bar, 50 µm. d. Quantification of panel b, where 100-600 cells for each condition were quantitated over one screening experiment with multiple fields of view. The exact number of cells for each condition are listed in the Source Data for Extended Data Fig. 8. e. Immunostaining of Oct4 after activation by paired crRNA consisting of the two most potent crRNAs (O1 + O2), which shows lack of additive effect. White scale bar, 100 µm. f. Quantification in panels e, where 200-700 cells for each condition were quantitated over one screening experiment with multiple fields of view. For box-and-whisker plots, the box shows 25-75% (with bar at median, dot at mean), and whiskers encompass 10-90%, with individual data points shown for the lowest and highest 10% of each dataset. The exact number of cells for each condition is listed in the Source Data for Extended Data Fig. 8.

Source data

Extended Data Fig. 9 Enhanced multiplex activation by hyperdCas12a.

Multiplex endogenous gene activation by hyperdCas12a versus enAsdCas12a and 6-crRNA array targeting Oct4, Sox2 and Klf4 (OSK) in mouse P19 cells as measured by qPCR, in similar experiment as described in Fig. 3a. Each data point shows one independent measurement, and each bar shows the average of n = 3 independent experiments. DR, direct repeat; NT, non-targeting.

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Extended Data Fig. 10 In vivo single crRNA activation by hyperdCas12a.

a-c, Constructs containing hyperdCas12a and single crRNA to Sox2 (a), Klf4 (b) or Oct4 (c) for in vivo electroporation in post-natal mouse retina and representative immunofluorescence images. CAG-GFP is used to mark the electroporated patch. Scale bar, 50 µm. Magnified views of the regions in the yellow boxes are shown in Fig. 6a. d, Quantification of percentages of Oct4+, Sox2+ or Klf4+ cells among HA + cells in INL. Bar graph shows the mean of 3 independent experiments, and each data point represents value of an independent experiment. e, Immunofluorescence images of in vivo electroporation in mouse retina with hyperdCas12a with non-targeting LacZ crRNA. Scale bar, 50 µm.

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Supplementary information

Reporting Summary

Supplementary Tables 1–4

Supplementary Table 1. crRNA sequences. Supplementary Table 2. Plasmids used in each figure. Supplementary Table 3. Primer sequences. Supplementary Table 4. All genes from RNA-seq.

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Guo, L.Y., Bian, J., Davis, A.E. et al. Multiplexed genome regulation in vivo with hyper-efficient Cas12a. Nat Cell Biol 24, 590–600 (2022). https://doi.org/10.1038/s41556-022-00870-7

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