Abstract
CRISPR–Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein–guide RNA complex3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38–58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5′ and 3′ untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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Data availability
Plasmids encoding the most relevant constructs shown in this work, including both SECURE BE3 variants, have been deposited to Addgene (http://www.addgene.org/browse/article/28197497/; Addgene IDs 123611–123616).
All RNA-seq data used in this study have been deposited in the Gene Expression Omnibus (GEO) repository (National Center for Biotechnology Information). The files are accessible through the GEO Series accession number GSE121668. All WES and targeted amplicon sequencing data have been deposited at the SRA repository under bioproject number PRJNA497753. All other relevant data are available from the corresponding author on request.
Code availability
The authors will make all previously unreported custom computer code used in this work available upon reasonable request.
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Acknowledgements
J.K.J., J.G. and R.Z. are supported by the Defense Advanced Research Projects Agency (HR0011-17-2-0042). Support was also provided by the National Institutes of Health (RM1 HG009490 to J.K.J. and J.G. and R35 GM118158 to J.K.J. and M.J.A.). J.K.J. is additionally supported by the Desmond and Ann Heathwood MGH Research Scholar Award. We thank M. M. Kaminski, B. P. Kleinstiver and K. Petri for discussions; V. Pattanayak for input on the manuscript; Y. E. Tak, G. Boulay, M. K. Clement, A. A. Sousa, R. T. Walton, M. L. Bobbin, M. V. Maus and A. Schmidts for technical advice; and P. K. Cabeceiras and O. R. Cervantes for technical assistance. J.K.J. dedicates this paper to the memory of C. J. Park.
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J.G. and R.Z. performed all wet laboratory experiments together. S.P.G., S.I., C.A.L. and M.J.A. performed all bioinformatic and computational analysis of data. J.G. and J.K.J. conceived and designed the study. J.G., M.J.A. and J.K.J. organized and supervised the work. J.G. and J.K.J. wrote the initial draft of the manuscript and all authors contributed to the writing of the final manuscript.
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Competing interests
J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Endcadia, Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. These interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. and M.J.A. also hold equity in Excelsior Genomics. J.K.J. is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.G., R.Z. and J.K.J. are co-inventors on patent applications that have been filed by Partners Healthcare/Massachusetts General Hospital on engineered base editor architectures that reduce RNA editing activities.
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Extended data figures and tables
Extended Data Fig. 1 Additional data and analysis for transcriptome-wide off-target C-to-U RNA editing induced with BE3 in HepG2 cells.
a, Dot plot of RNF2 on-target DNA editing data shown in Fig. 1b, depicting editing frequencies for all cytosines across the spacer sequence. b, Heat maps showing RNA and DNA editing efficiencies with BE3 and control on cytosines in human APOB. Numbering indicates nucleotide positions in the APOB transcript; asterisks identify those previously shown to be modified by APOBEC1. c, Histograms showing numbers of RNA-edited cytosines identified (y-axis) with RNA C-to-U editing frequencies (x-axis) for the four replicates shown in Fig. 1c. Dashed red line, median; solid red line, mean. d, Manhattan plots of data for replicates 2, 3, and 4 from Fig. 1c showing the distribution of modified cytosines identified across the transcriptome. n, total number of modified cytosines identified. e, Percentages of different predicted effects and locations of edited cytosines identified in each RNA-seq replicate. f, Jitter plots of cytosines modified by BE3 expression with the RNF2 gRNA categorized by their presence in 4, 3, 2 or 1 of the replicate RNA-seq experiments performed in HepG2 cells (n = 4 biologically independent samples, as in Fig. 1c). Box spans the interquartile range (IQR) (first to third quartiles); horizontal line shows median (second quartile); whiskers extend to ± 1.5 × IQR. n, total number of modified cytosines present in each category. The percentage of all modified cytosines in each category is also shown.
Extended Data Fig. 2 BE3 expression with two different gRNAs induces transcriptome-wide off-target RNA editing in HEK293T cells.
a, Heat maps of on-target DNA base editing efficiencies of BE3 and nCas9–UGI–NLS (control) in HEK293T cells (all GFP sorting) determined in triplicate with RNF2 or EMX1 gRNA. Bases shown are within the editing window of the on-target spacer sequence (numbering is at the bottom with 1 being the most PAM-distal spacer position). b, Dot plots of RNF2 and EMX1 on-target DNA editing data shown in a, depicting editing frequencies for all cytosines across the spacer sequence. c, Jitter plots derived from RNA-seq experiments showing RNA cytosines modified by BE3 expression with RNF2 or EMX1 gRNA. n, total number of modified cytosines identified in each replicate. d, Histograms showing numbers of RNA-edited cytosines identified (y-axis) with RNA C-to-U editing frequencies (x-axis) for the experiments shown in c. Dashed red line, median; solid red line, mean. e, Manhattan plots of data shown in c depicting the distribution of modified cytosines across the transcriptome. n, total number of modified cytosines identified.
Extended Data Fig. 3 Additional analysis of data showing transcriptome-wide off-target RNA editing in HEK293T cells with BE3 and two different gRNAs.
a, Percentages of different predicted effects and locations of edited cytosines in each RNA-seq replicate from Extended Data Fig. 2c. b, Percentages (x-axis) and numbers (shown inside bars) of expressed genes in each RNA-seq replicate from data shown in Extended Data Fig. 2c that show at least one edited cytosine. c, Jitter plots of cytosines modified by BE3 expression with RNF2 or EMX1 gRNA categorized by their presence in 3, 2 or 1 of the replicate RNA-seq experiments performed in HEK293T cells (n = 3 biologically independent samples, as in Extended Data Fig. 2c). Box, whiskers and n are as defined in Extended Data Fig. 1f. The percentage of all modified cytosines identified in each category is also shown. d, Sequence logos derived from edited cytosines identified in each RNA-seq replicate. Analysis done using RNA-seq data generated from cDNA; every T depicted should be considered a U in RNA. e, Venn diagram showing numbers of cytosines edited with the RNF2 and EMX1 gRNAs. For each gRNA, the number of cytosines represents the union of those identified in the three replicates.
Extended Data Fig. 4 Increased BE3 expression induces higher numbers and frequencies of transcriptome-wide RNA cytosine edits in HEK293T cells.
a, Heat maps of on-target DNA base editing efficiencies of BE3 and nCas9–UGI–NLS (control) in HEK293T cells (top 5% GFP sorting) determined in duplicate with RNF2 or EMX1 gRNA. Bases shown are within the editing window of the on-target spacer sequence (numbering is at the bottom with 1 being the most PAM-distal spacer position). b, Dot plots of data shown in a, depicting editing frequencies for all cytosines across the spacer sequence. c, Jitter plots derived from duplicate RNA-seq experiments showing RNA cytosines modified by BE3 expression with RNF2, EMX1 or non-targeted (NT) gRNA. n, total number of modified cytosines identified in each replicate. d, Histograms showing numbers of RNA edited cytosines identified (y-axis) with RNA C-to-U editing frequencies (x-axis) for the experiments shown in c. Dashed red line, median; solid red line, mean. e, Manhattan plots of data for both replicates of RNF2, EMX1, and non-targeted gRNAs from c showing the distribution of modified cytosines across the transcriptome. n, total number of modified cytosines identified.
Extended Data Fig. 5 Additional data and analysis showing that increased BE3 expression induces higher numbers and frequencies of transcriptome-wide RNA cytosine edits in HEK293T cells.
a, Percentages of different predicted effects and locations of edited cytosines identified in each RNA-seq replicate from Extended Data Fig. 4c. b, Percentages (x-axis) and numbers (shown inside bars) of expressed genes in each RNA-seq replicate that have at least one edited cytosine. c, Sequence logos derived from edited cytosines identified in each RNA-seq duplicate experiment from Extended Data Fig. 4c for the RNF2, EMX1 and non-targeted gRNAs. Analysis done using RNA-seq data generated from cDNA; every T depicted should be considered a U in RNA. d, Venn diagram showing numbers of edited cytosines identified with the RNF2, EMX1 and non-targeted gRNAs. For each gRNA, the circle encompasses the union of cytosines identified in the two replicates (data derived from the experiments shown in Extended Data Fig. 4c). e, Venn diagrams showing all possible pairwise comparisons of edited cytosines identified in duplicate experiments performed with the RNF2, EMX1 and non-targeted gRNAs (data derived from the experiments shown in Extended Data Fig. 4c). f, Scatter plot correlating RNA editing frequencies (x-axis) of 154,264 cytosines previously shown to be edited by RNA-seq with DNA editing frequencies (y-axis) determined by WES performed with DNA derived from the same experiments (n = 3 biologically independent samples, pooled data). Superimposed histograms (top and right) depict the percentages of cytosines that show various editing rates on RNA (upper x-axis) or DNA (right y-axis).
Extended Data Fig. 6 Additional data showing that SECURE BE3 variants induce substantially reduced numbers of RNA edits but possess comparable and more-precise DNA editing activities in HEK293T cells.
a, Initial screen of transcriptome-wide RNA editing activities of six BE3 variants containing various rAPOBEC1 mutations and expressed at high levels in HEK293T cells (sorting cells with top 5% of GFP signal). Jitter plots of single replicate RNA-seq experiments showing RNA cytosines modified by expression of wild-type BE3, BE3-E63Q (rAPOBEC1 catalytic site mutant), BE3-P29F, BE3-P29T, BE3-L182A, BE3-R33A, BE3-K34A and BE3-R33A/K34A variants. n, total number of modified cytosines identified in each sample. b, Heat map of on-target DNA base-editing efficiencies of nCas9–UGI–NLS (control), wild-type BE3, BE3-R33A and BE3-R33A/K34A in HEK293T cells with the RNF2 gRNA (cells from experiment shown in Fig. 2a). Bases within the editing window of the on-target spacer sequence are numbered as previously described. Note the inclusion of C12, which is inefficiently edited by wild-type BE3 in these samples but not edited by the SECURE BE3 variants, even with higher expression. c, Dot plot for HEK293T on-target data displayed in b, expanded to include all cytosines across the spacer sequence.
Extended Data Fig. 7 Additional data and analysis of the on-target DNA and off-target RNA activities of BE3 and SECURE BE3 variants.
a, Dot plots illustrating on-target DNA editing efficiencies of nCas9–UGI–NLS (control), wild-type BE3, BE3-R33A and BE3-R33A/K34A in HEK293T cells on 12 genomic sites. These are the same data as shown in Fig. 2c, expanded to include all cytosines across the spacer sequence. b, Jitter plots from RNA-seq experiments in HepG2 cells showing RNA cytosines modified by wild-type BE3, BE3-R33A and BE3-R33A/K34A. Data for wild-type BE3 are from the experiments presented in Fig. 1c (replicates 2–4). n, total number of modified cytosines identified. c, Manhattan plots of data showing the distribution of modified cytosines induced with BE3-R33A or BE3-R33A/K34A expression for replicate 3 from b overlaid on modified cytosines induced with wild-type BE3 expression (the wild-type BE3 data are the same in the top and bottom plots). n, total number of modified cytosines identified. d, Heat map of on-target DNA base editing efficiencies of nCas9–UGI–NLS (control), wild-type BE3, BE3-R33A and BE3-R33A/K34A in HepG2 cells with the RNF2 gRNA (cells from same experiment as shown in b). Replicates 1, 2 and 3 for wild-type BE3 and nCas9–UGI–NLS show the same data presented as replicates 2, 3 and 4 for wild-type BE3 and nCas9–UGI–NLS in Fig. 1b. Bases within the editing window of the on-target spacer sequence are numbered as previously described. Note again the inclusion of position C12. e, Dot plot for HepG2 on-target data shown in d, expanded to include all cytosines across the spacer sequence. f, Schematic of the editing windows (coloured boxes) for wild-type BE3, BE3-R33A and BE3-R33A/K34A based on experimental data from Fig. 2c and Extended Data Fig. 7a. Darker-coloured and more-translucent boxes indicate positions generally showing higher and lower C-to-T editing efficiencies, respectively. Increased stringency for a 5′T with BE3-R33A/K34A is also indicated. The PAM (NGG) and the nicking site in the DNA backbone are highlighted. Drawings are adapted with permission from table 1 of ref. 1.
Extended Data Fig. 8 Additional data and analysis for transcriptome-wide off-target A-to-I RNA editing induced by ABEmax expression in HEK293T cells.
a, Dot plot of HEK site 2 on-target DNA editing data shown in Fig. 3a, depicting editing frequencies for all adenines across the spacer sequence. b, Histograms showing numbers of RNA-edited adenines identified (y-axis) with RNA A-to-I editing frequencies (x-axis) for three replicates shown in Fig. 3b. Dashed red line, median; solid red line, mean. c, Manhattan plots of data for replicates 1 and 2 from Fig. 3b showing the distribution of modified adenines identified across the transcriptome. n, total number of modified adenines identified. d, Percentages of different predicted effects and locations of edited adenines in each RNA-seq replicate shown in Fig. 3b. e, Percentages (x-axis) and numbers (inside bars) of expressed genes in each RNA-seq replicate that show at least one edited adenine. f, Jitter plots of adenines modified by ABEmax expression with the HEK site 2 gRNA categorized by their presence in 3, 2 or 1 of the replicate RNA-seq experiments shown in Fig. 3b (n = 3 biologically independent samples). Box and whiskers are as defined in Extended Data Fig. 1f. n, total number of modified adenines present in each category. The percentage of all modified adenines found in each category is also shown. g, Scatter plot correlating RNA editing frequencies (x-axis) of 52,462 adenines previously shown to be RNA edited with DNA editing frequencies (y-axis) determined by WES (n = 3 biologically independent samples, pooled data). Superimposed histograms (top and right) depict the percentages of edited adenines on RNA (upper x-axis) or DNA (right y-axis).
Extended Data Fig. 9 Effects of BE3 and SECURE BE3 variants on cell viability, structural model of rAPOBEC1 and extended sequence logos of off-target RNA edited sites.
a, Cell viability assay comparing HEK293T cells transfected with plasmid expressing nCas9–UGI–NLS, wild-type (WT) BE3, BE3-R33A, BE3-R33A/K34A or BE3-E63Q (n = 3 biologically independent samples per condition). Each dot represents one biological replicate (and is the mean of three technical replicates). All data points were normalized to the mean luminescence of the nCas9–UGI–NLS controls (set to 100%, grey dotted line) that were performed for each biological replicate experiment. The assay was performed on days 1, 2, 3 and 4 after plating (following sorting for all GFP-positive cells). Data shown as mean ± s.e.m. RLU, relative light unit; n.s., not significantly decreased compared to matched nCas9–UGI–NLS control; *P < 0.05, ***P < 0.001 for a significant decrease compared to matched nCas9–UGI–NLS control. Statistical significance was determined as described in Supplementary Methods. b, Structural model of rAPOBEC1 with locations of catalytic residues and the R33 and K34 positions that were altered in SECURE variants. A predicted rAPOBEC1 structure is shown that was generated with Protein Homology/analogY Recognition Engine v 2.0 (Phyre2)37 and visualized in PyMOL (v 1.8.2.1). The R33 and K34 residues mutated in the SECURE variants are shown in orange and blue, respectively. Catalytic site residues (H61, E63, C93 and C96) have previously been described19 and are shown in green. c–f, Extended sequence logos for BE3- and ABEmax-induced RNA editing sites. Sequence logos derived with the nucleotides 100 base pairs upstream and downstream of the motifs edited in RNA by BE3 (ACW) or ABEmax (UA) are shown. Logos were derived from data for BE3 expression in HepG2 cells (c; Fig. 1c), BE3 expression in HEK293T cells (d; all GFP-sorted cells; Extended Data Fig. 2c), higher BE3 expression in HEK293T cells (e; top-5% GFP-sorted cells; Extended Data Fig. 4c), and ABEmax expression in HEK293T experiments (f; top 5% GFP-sorted cells; Fig. 3b). Analysis was done using RNA-seq data generated from cDNA; every T depicted should be considered a U in RNA.
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Grünewald, J., Zhou, R., Garcia, S.P. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019). https://doi.org/10.1038/s41586-019-1161-z
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DOI: https://doi.org/10.1038/s41586-019-1161-z
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