Data availability
All source data (including P values) are available in Supplementary Table 5. Raw FASTQ files have been deposited in the Sequence Read Archive under accession number PRJNA859154. Processed RNA-seq data (transcripts per million values and differentially expressed genes) are available in Supplementary Table 2.
Code availability
Code for analysis and data visualization is available at https://github.com/lukedow/iBE.git.
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Acknowledgements
We thank members of the Dow laboratory for advice and comments on the preparation of the paper. We would like to acknowledge K. Tsanov and J. Leibold for assistance and advice in setting up the pancreas EPO protocol. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH). This work was supported by a project grant from the NIH (R01CA229773), P01 CA087497 (S.W.L.), an MSKCC Functional Genomics Initiative grant (S.W.L.), an Agilent Technologies Thought Leader Award (S.W.L.) and support from Synthego under a Synthego Innovator Award (L.E.D.). A.K. was supported by an F31 Ruth L. Kirschstein Predoctoral Individual National Research Service Award (F31-CA247351-02). A.V. was supported by a Postdoctoral Fellowship from the Human Frontier Scientific Program (LT0011/2023-L). B.J.D. was supported by an F31 Ruth L. Kirschstein Predoctoral Individual National Research Service Award (F31-CA261061-01). E.E.G. is the Kenneth G. and Elaine A. Langone Fellow of the Damon Runyon Cancer Research Foundation (DRG-2343-18). F.J.S.R. was supported by the MSKCC TROT program (5T32CA160001) and a GMTEC Postdoctoral Researcher Innovation Grant and is a Howard Hughes Medical Institute (HHMI) Hanna Gray Fellow. S.W.L. is an HHMI investigator.
Author information
Author notes
Miguel Foronda
Present address: Memorial Sloan Kettering Cancer Center, New York, NY, USA
Maria Paz Zafra
Present address: Biosanitary Research Institute (IBS)–Granada, Granada, Spain
Francisco J. Sánchez Rivera
Present address: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
Francisco J. Sánchez Rivera
Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
These authors contributed equally: Alyna Katti, Adrián Vega-Pérez.
Authors and Affiliations
Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
Alyna Katti, Adrián Vega-Pérez, Miguel Foronda, Jill Zimmerman, Maria Paz Zafra, Elizabeth Granowsky, Sukanya Goswami, Eric E. Gardner, Bianca J. Diaz, Maria Teresa Calvo Fernandez & Lukas E. Dow
Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA
Alyna Katti, Jill Zimmerman, Bianca J. Diaz & Lukas E. Dow
Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Janelle M. Simon, Alexandra Wuest, Wei Luan, Scott W. Lowe & Francisco J. Sánchez Rivera
Synthego Corporation, Redwood City, CA, USA
Anastasia P. Kadina, John A. Walker II & Kevin Holden
Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Scott W. Lowe
Department of Medicine, Weill Cornell Medicine, New York, NY, USA
Lukas E. Dow
Contributions
A.K. and A.V.P. designed and performed experiments, analyzed data and wrote the paper. M.F., J.Z., M.P.Z., S.G., E.G., J.S., W.L, B.J.D., M.C.F., K.H. and F.J.S.R. performed experiments and/or analyzed data. S.W.L. supervised experimental work. L.E.D. designed and supervised experiments, analyzed data and wrote the paper.
Corresponding author
Ethics declarations
Competing interests
L.E.D. is a scientific advisor and holds equity in Mirimus, Inc. L.E.D. has received consulting fees and/or honoraria from Volastra Therapeutics, Revolution Medicines, Repare Therapeutics, Fog Pharma and Frazier Healthcare Partners. S.W.L is an advisor for and has equity in the following biotechnology companies: ORIC Pharmaceuticals, Faeth Therapeutics, Blueprint Medicines, Geras Bio, Mirimus, Inc., PMV Pharmaceuticals and Constellation Pharmaceuticals. S.W.L. acknowledges receiving funding and research support from Agilent Technologies for the purposes of massively parallel oligo synthesis. K.H., A.P.K. and J.A.W. are employees and shareholders of Synthego Corporation.
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Extended data
Extended Data Fig. 1 Regulatable BE expression in vivo.
a. Calculated BE3RA transgene copy number in iBEhem and iBEhom using a Taqman quantiative PCR assay with genomic DNA from H11-LSL-Cas9 mice as a reference. Data are presented as mean values ± s.e.m. (*pother SNVs found in pooled clones for each condition (on and off dox) for both sgRNAs. c. Sequencing analysis at cancer gene sites in cell conditions (right) described in a. Solid blue boxes represent on-target activity of the sgRNA, dotted orange boxes signify on-target ‘bystander’ editing within the gRNA window. d. Quantification of C>T and C>other SNVs found across both targets. 2-way ANOVA test for multiple comparisons was used to evaluate statistical significance across conditions. Data are presented as mean values ± s.e.m. p-values are displayed.
Extended Data Fig. 6 iBE does not induce off target RNA editing in organoids.
a. Schematic of experimental set up in iBE derived pancreatic organoids. Organoids were transduced and selected with GFPGO reporter (mScarlet+). Organoids maintained off dox were then split into dox conditions to induce BE expression for 4 days and then split again into + and – dox conditions for an additional day. b. Editing of organoids in each condition (OFF, D4, D8, and D4 sw) was quantified by flow cytometry, calculating the percentage of GFP+ cells within the mScarlet+ population. Data are presented as mean values ± s.e.m. One-way ANOVA with Tukey’s correction c. PCA analysis of RNA sequencing data from OFF, D8, and D4 SW organoids. Colors correspond to dox condition and shape delineates organoid replicate/mouse origin (n=3). d. Volcano plots from RNA-seq data comparing iBE pancreatic organoids culture on dox-containing media vs regular media. e. Off-target RNA editing analysis, processed as described for Supplementary Fig. 4. No significant differences in RNA variants were observed, n=3, one-way ANOVA with Tukey’s correction. Data are presented as mean values ± s.e.m. For all data shown, n=3 independent organoid lines/condition.
Extended Data Fig. 7 Editing dynamics of iBE organoids.
a. Flow cytometry analysis of three independent pancreatic KP mutant organoid lines integrated with GFPGO reporter following dox treatment for 0-8 days (black), transient exposure for 2h or 12h (grey), or transient exposure then re-treatment at 4 days (green). b. Targeted deep sequencing quantification of target C:G to T:A conversion at the ApcQ1405X locus in 2D small intestinal derived iBE cell line following dox addition for 21 days (dark blue), or transient dox treatment for 3 days and withdrawn for 18 days (light blue). c. Targeted deep sequencing quantification of indel conversion of b. Data are presented as mean values ± s.e.m. (*pT/A/G and indel conversion in small intestinal iBE organoids nucleofected with plasmid (light blue) or synthetic (indigo) gRNAs (ApcQ1405, Trp53Q97, CR8.OS2) as indicated, with and without dox treatment. b. Targeted deep sequencing quantification of target C>T/A/G and indel conversion in small intestinal iBE organoids nucleofected with synthetic gRNAs targeting cancer associated SNVs from Fig. 2f. c-j. Quantification of collateral editing of adjacent cytosines for samples shown in Fig. 2f. Predicted translation of each quantified read is shown below with targeted amino acid substitution (dark grey) and additional amino acid substitution (pink). All data are presented as mean values ± s.e.m.
Extended Data Fig. 9 Analysis of collateral editing before and after functional selection.
a-h. Quantification of collateral editing of adjacent cytosines for data shown in Fig. 2f, unselected (white) and selected (color) in small intestinal iBE organoids nucleofected with various synthetic gRNAs targeting cancer associated SNVs.
Extended Data Fig. 10 In situ base editing with iBE by synthetic gRNA delivery drives liver tumors.
a. HTVI delivery of synthetic gRNAs with SB-Myc as in Fig. 4. BF, H&E images, and IF staining for ß-catenin (green) and glutamine synthetase (GS, red) in livers with tumors. Number of transfected mice with palpable tumors is shown below each column. b. Quantification of target C:G to T:A conversion from tumors described in a). Each point corresponds to an isolated bulk tumor. (n=2-7 mice for a given gRNA target). Individual editing data color-coded by animal in Supplementary Fig. 3. All data are presented as mean values ± s.e.m.
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Katti, A., Vega-Pérez, A., Foronda, M. et al. Generation of precision preclinical cancer models using regulated in vivo base editing.
Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01900-x
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Received: 14 July 2022
Accepted: 10 July 2023
Published: 10 August 2023
DOI: https://doi.org/10.1038/s41587-023-01900-x
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