Abstract
CRISPR–Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.
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Data availability
Sequencing data are available in the Sequence Read Archive under accession number PRJNA879077. Source data are provided with this paper. Data for supplementary figures are available as supplementary datasets.
Code availability
Scripts written for parsing data and plotting figures are available on Github (https://github.com/cindytxwei/ciCas9effectors). Scripts written for the permutation analysis based on the chi-squared test statistic are also available on Github (https://github.com/omripel/BEAnalysis).
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Acknowledgements
This work was supported by the NIH, grant nos. RM1HG010461 (NHGRI) and R01GM109110 (NIGMS) to D.M.F., and grant no. R01GM145011 (NIGMS) to D.J.M. O.P. was supported in part by a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel Aviv University. E.B. is a Faculty Fellow of the Edmond J. Safra Center for Bioinformatics at Tel Aviv University.
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Contributions
C.T.W., D.J.M. and D.M.F. conceived the work and wrote the paper. C.T.W. performed all base and prime editing experiments and data analysis. C.T.W. performed the transcriptional activation experiments with dciCas9-VPR in Fig. 1 and Supplementary Fig. 3. N.A.P. and R.L.P. performed the transcriptional activation experiments with dciCas9-VPR in Extended Data Fig. 1. O.P. and E.B. designed and performed the chi-squared test for the base editing dependency analysis.
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C.T.W. is a current employee at the Novartis Institutes for BioMedical Research Inc.
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Nature Chemical Biology thanks Chase Beisel 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 Transcriptional activation of a diverse range of target sequences using dciCas9-VPR.
Activation of an EGFP reporter locus downstream of the indicated target sequence using dCas9-VPR or dciCas9-VPR targeted to this synthetic locus (see Methods) in the presence or absence of 1 μM A115. Cells were treated with A115 for 48 hr prior to flow cytometry analysis. Bars represent the geometric mean EGFP fluorescence ± SEM of three cell culture replicates. Three cell culture replicates are shown in the blue circles overlapping each bar.
Extended Data Fig. 2 Chemically-controlled cytidine base editors without codon optimization.
a) Schematic of the domain arrangements in the unmodified BE4max and AncBE4max base editors and the chemically-controlled BE4max and AncBE4max base editors without codon optimization and using the ciCas9(L22) variant. 3 different versions of ciCas9 were used, ciCas9(L22), ciCas9(L22) without a Flag-tag (ΔFlag), and ciCas9(L22) without a Flag-tag and additional SV40-NLS (ΔFlag/ΔNLS). b-c) C-to-T editing frequency with BE4max and BE4max-ciCas9 at the EMX1 (b) and HEK3 (c) target sites. d-e) C-to-T editing frequency with AncBE4max and AncBE4max-ciCas9 at the EMX1 (d) and HEK3 (e) target sites. BE4max and AncBE4max editing were measured at 48 and 72 hr after co-transfection of BE4max and sgRNA. BE4max-ciCas9 and AncBE4max-ciCas9 editing were measured at 24 and 72 hr after 1 μM A115 addition. C-to-T editing is shown at the 2 nucleotides in each target site with highest editing frequency with the Cas9 version of base editors (BE4max or AncBE4max). The 2 different nucleotides are indicated by color in the target sequence. Bars show mean editing frequency ± SEM of 3 cell culture replicates with white circles showing individual replicates.
Extended Data Fig. 3 Heatmaps of base editing by chemically-controlled base editors compared to unmodified base editors.
a–b) Heatmaps of BE4max, ciBE4max (a) and AncBE4max, ciAncBE4max (b) C-to-T base editing as a percentage of the highest edited nucleotide for each editor throughout the entire indicated target sites. c-d) Heatmaps of ABEmax, ciABEmax (c) and ABE8e, ciABE8e (d) A-to-G base editing as a percentage of the highest edited nucleotide for each editor throughout the entire indicated target sites. Editing is represented as a percentage of the highest edited nucleotide to allow comparison of the spatial distribution of edits between base editors. Each row shows an individual cell culture replicate. Editing frequencies of the unmodified base editors were quantified at 72 hr after transfection for the HEK3 target site and 48 hr after transfection for the ABE9 and HEK2 target sites. Chemically-controlled base editing frequencies were quantified at 72 hr after 1 μM A115 addition to HEK293T cells for the HEK3 target site and 24 hr after 1 μM A115 addition to HEK293T cells for the ABE9 and HEK2 target sites. The control shows untransfected cells harvested at the same time as the chemically-controlled base editors. The numbers below the heatmaps show the position of the nucleotide from the most PAM-distal nucleotide.
Extended Data Fig. 4 Early time points in time courses of base editing with the chemically-controlled base editors.
Early time courses of chemically-controlled base editing using ciBE4max (a), ciABEmax (b), and ciABE8e (c) activated using 1 μM A115 at the indicated target sites. Time courses shown for the nucleotide colored in the target sequences shown. Numbers underneath the target sequence show the position of the nucleotide from the most PAM-distal nucleotide. Bars show mean editing ± SEM of 3 cell culture replicates with white circles showing individual replicates. Significance of editing at different time points were compared to editing frequency at 0 hr using a One-way ANOVA, statistical values shown in Supplementary Table 2. In (a), **P = 0.0033, ***P = 0.0005, ****P = < 0.0001. In (b), ****P = < 0.0001, ABE9 ***P = 0.0003, ABE16 ***P = 0.0002. In (c), ****P = < 0.0001, ***P = 0.0007, ABE16 *P = 0.0315, HEK3 *P = 0.0228.
Extended Data Fig. 5 Time courses of base editing with the chemically-controlled base editors.
a) Time course of chemically-controlled cytidine base editing by ciBE4max at the ABE9, EMX1, HEK2, and HEK3 target sites. ciBE4max was activated with 1 μM A115. Cells were harvested and editing was quantified at specified time points after activation. Colors of lines represent the corresponding nucleotide within the target site. Numbers underneath the target sequence show the position of the nucleotide from the most PAM-distal nucleotide. b,c) Time course of chemically-controlled adenine base editing by ciABEmax (b) and ciABE8e (c) at the ABE9, ABE16, HEK2, and HEK3 target sites. ciABEmax and ciABE8e were activated with 1 μM A115. Cells were harvested and editing was quantified at specified time points after activation. Colors of lines represent the corresponding nucleotide within the target site. Numbers underneath the target sequence show the position of the nucleotide from the most PAM-distal nucleotide. Data represented as mean editing ± SEM of 3 cell culture replicates. Time courses shown for all nucleotides where base editing frequency was greater than 0.5% at 24 hr after A115 addition.
Extended Data Fig. 6 Time courses of ciBE4max base editing allele outcomes.
Time course of allele formation by ciBE4max after activation with 1 μM A115 or DMSO. Black lines and circles show editing with 1 μM A115, gray lines and circles show editing with DMSO. Data represented as mean allele frequency ± SEM of 3 cell culture replicates.
Extended Data Fig. 7 Time courses of ciABE8e base editing allele outcomes.
Time course of allele formation by ciABE8e after activation with 1 μM A115 or DMSO. Black lines and circles show editing with 1 μM A115, gray lines and circles show editing with DMSO. Data represented as mean allele frequency ± SEM of 3 cell culture replicates.
Extended Data Fig. 8 Time course of measured and expected allele frequencies by ciBE4max.
Measured and expected allele frequencies over time created by ciBE4max that show a dependent model of base editing for multiply-edited alleles. Black lines and solid circles show measured allele frequencies, gray lines and open circles show expected allele frequencies. Measured data represented as mean editing frequency ± SEM of 3 cell culture replicates. Expected editing frequency represented as mean expected editing frequency ± relative error. Calculations for expected frequency and relative error described in the methods.
Extended Data Fig. 9 Time course of measured and expected allele frequencies by ciABE8e.
Measured and expected allele frequencies over time created by ciABE8e that show a dependent model of base editing for multiply-edited alleles. Black lines and solid circles show measured allele frequencies, gray lines and open circles show expected allele frequencies. Measured data represented as mean editing frequency ± SEM of 3 cell culture replicates. Expected editing frequency represented as mean expected editing frequency ± relative error. Calculations for expected frequency and relative error described in the Methods.
Extended Data Fig. 10 Heatmaps of SPACE and ciSPACE base editing.
Heatmaps of SPACE and ciSPACE editing through the entire HEK2 (top) and HEK3 (bottom) target sites. A-to-G base editing is shown in pink, C-to-T base editing is shown in blue. Editing is shown as a percentage of the highest edited nucleotide for each editor for that target site. Editing is represented as a percentage of the highest edited nucleotide to allow comparison of the spatial distribution of edits between base editors. Each row shows an individual cell culture replicate. SPACE editing frequencies were quantified at 72 hr after transfection and ciSPACE editing frequencies were quantified at 72 hr after 1 μM A115 addition to HEK293T cells. The control shows untransfected cells harvested at the same time as ciSPACE. The numbers below the heatmaps show the position of the nucleotide from the most PAM-distal nucleotide.
Supplementary information
Supplementary Information (download PDF )
Supplementary Figs. 1–22 and Tables 1–7.
Supplementary Data (download XLSX )
Supplementary Datasets 1–15 corresponding to the Supplementary Figs.
Source data
Source Data Fig. 1 (download XLSX )
Median fluorescence values from flow cytometry.
Source Data Fig. 2 (download XLSX )
Base editing frequencies for cytidine base editors.
Source Data Fig. 3 (download XLSX )
Base editing frequencies for adenine base editors.
Source Data Fig. 4 (download XLSX )
Base editing frequencies for time courses.
Source Data Fig. 5 (download XLSX )
Base editing allele frequencies for time courses.
Source Data Fig. 6 (download XLSX )
Base editing frequencies for SPACE/ciSPACE, prime editing frequencies for PE2/ciPE2.
Source Data Extended Data Fig. 1 (download XLSX )
Median fluorescence values from flow cytometry, additional target sites.
Source Data Extended Data Fig. 2 (download XLSX )
Base editing frequencies for non-codon-optimized cytidine base editors.
Source Data Extended Data Fig. 3 (download XLSX )
Heat maps of base editing frequencies by unmodified and chemically controlled base editors.
Source Data Extended Data Fig. 4 (download XLSX )
Base editing frequencies at early time points for chemically controlled base editors.
Source Data Extended Data Fig. 5 (download XLSX )
Base editing frequencies for full time courses.
Source Data Extended Data Fig. 6 (download XLSX )
Base editing allele frequencies for ciBE4max.
Source Data Extended Data Fig. 7 (download XLSX )
Base editing allele frequencies for ciABE8e.
Source Data Extended Data Fig. 8 (download XLSX )
Measured and expected allele frequency time courses for ciBE4max.
Source Data Extended Data Fig. 9 (download XLSX )
Measured and expected allele frequency time courses for ciABE8e.
Source Data Extended Data Fig. 10 (download XLSX )
Heat maps of base editing frequencies by unmodified and chemically controlled SPACE.
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Wei, C.T., Popp, N.A., Peleg, O. et al. A chemically controlled Cas9 switch enables temporal modulation of diverse effectors. Nat Chem Biol 19, 981–991 (2023). https://doi.org/10.1038/s41589-023-01278-6
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DOI: https://doi.org/10.1038/s41589-023-01278-6
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