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CRISPR DNA base editors with reduced RNA off-target and self-editing activities

Nature Biotechnology volume 37, pages 1041–1048 (2019)Cite this article

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Abstract

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1,2,3,4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.

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Fig. 1: Engineering of SECURE-ABE variants with reduced off-target RNA-editing activity.
Fig. 2: On-target DNA-editing activity of ABEmax, miniABEmax(K20A/R21A) and miniABEmax(V82G) in HEK293T cells.
Fig. 3: Transcriptome-wide off-target RNA-editing activity of CBEs with non-APOBEC1 cytidine deaminases in HEK293T cells.
Fig. 4: Self-editing generates heterogeneously edited CBE and ABE transcript sequences in HEK293T and HepG2 cells.

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Data availability

Plasmids encoding the SECURE-ABE and various CBE constructs shown in this work are available on Addgene under article number 28203996. The RNA-seq data used in this study have been deposited in the Gene Expression Omnibus (GEO) under accession GSE129894.

Targeted amplicon sequencing data have been deposited at the Sequence Read Archive BioProject accession number PRJNA553185. 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 (grant HR0011-17-2-0042). Support was also provided by the National Institutes of Health (grant RM1 HG009490 to J.K.J. and J.G., and grant R35 GM118158 to J.K.J. and M.J.A.). J.G. was supported by a research fellowship (GR 5129/1-1) of the German Research Foundation (DFG). J.K.J. is additionally supported by the Desmond and Ann Heathwood MGH Research Scholar Award. We thank G. Ciaramella for the suggestion to delete the wild-type TadA monomer from ABEmax. We thank A. Lapinaite for suggesting the overlay of E. coli and S. aureus TadA structures and S.J. Lee for technical assistance.

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Author notes

  1. These authors contributed equally: Sowmya Iyer, Caleb A. Lareau, Sara P. Garcia.

Authors and Affiliations

  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA

    Julian Grünewald, Ronghao Zhou, Sowmya Iyer, Caleb A. Lareau, Sara P. Garcia, Martin J. Aryee & J. Keith Joung

  2. Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA

    Julian Grünewald, Ronghao Zhou, Martin J. Aryee & J. Keith Joung

  3. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Julian Grünewald, Martin J. Aryee & J. Keith Joung

  4. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Caleb A. Lareau & Martin J. Aryee

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  1. Julian Grünewald
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Contributions

All wet lab experiments were performed by R.Z. and J.G. S.I., C.A.L., S.P.G. and M.J.A. performed computational analysis of the data. J.G. and J.K.J. conceived of and designed the study. J.G., M.J.A. and J.K.J. supervised the work. J.G. and J.K.J. wrote the initial manuscript draft and all authors contributed to the writing of the final manuscript.

Corresponding author

Correspondence to J. Keith Joung.

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Competing interests

J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, Transposagen Biopharmaceuticals and Verve Therapeutics (f/k/a Endcadia). The interests of J.K.J. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. M.J.A. holds 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 and increase their precision.

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Integrated supplementary information

Supplementary Figure 1 On-target DNA editing and additional data on the off-target RNA editing activities of ABEmax, miniABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G in HEK293T cells.

(a) Heat maps showing the on-target DNA editing efficiencies of nCas9 (Control), ABEmax, miniABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G each assessed with two gRNAs targeted to HEK site 2 and ABE site 16 and performed in triplicate. Note that these were performed with genomic DNA isolated from cells that were sorted for the RNA-seq experiments shown in Fig. 1c, d. Editing windows shown include only the most highly edited adenines and not the entire spacer sequence. Numbering at the bottom represents spacer position with 1 being the most PAM distal location. (b) Histograms showing the total number of RNA A-to-I edits observed (y-axis) with different editing efficiencies (x-axis) for ABEmax, miniABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G each tested with the HEK site 2, ABE site 16, and NT gRNAs. n = number of modified adenines. Experiments were performed in triplicate (data is derived from the same experiments as Fig. 1c, d). Dashed line = median; solid line = mean. Rep. = Replicate.

Supplementary Figure 2 Sequence logos for RNA adenines edited by ABEmax, miniABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G in HEK293T cells.

Sequence logos derived using all RNA-edited adenines (0–100]% or stratified RNA-edited adenines with high (80–100]%, middle (50–80]%, or low (0–50]% editing efficiencies induced by (a) ABEmax co-expressed with a HEK site 2, ABE site 16, or NT (non-targeting) gRNA or (b) miniABEmax co-expressed with a HEK site 2, ABE site 16, or NT gRNA or (c) miniABEmax-K20A/R21A or miniABEmax-V82G co-expressed with a HEK site 2, ABE site 16, or NT gRNA. Logos are shown for triplicate experiments from the same RNA-seq experiments displayed in Fig. 1c, d. n = total number of modified adenines. For strata that contained <50 edited adenines, we considered the motif analysis as not sufficiently powered and therefore presented these logos in a semi-transparent fashion or using all adenines (i.e., (0–100]% editing efficiency).

Supplementary Figure 3 Unexpected on-target editing of C bases by ABEs in HEK293T cells.

Heat maps showing (a) C-to-G (pink), (b) C-to-T (purple), and (c) C-to-A (green) DNA on-target editing efficiencies induced by nCas9 (Control), ABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G each co-expressed with HEK site 2, ABE site 7, or FANCF site 1 gRNAs (n=4 independent replicates). These were obtained from the same data presented in Fig. 2a, b but now including the entire spacer sequence and showing these other types of base edits. Numbering at the bottom represents spacer position with 1 being the most PAM-distal location.

Supplementary Figure 4 DNA off-target activities of ABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G in HEK293T cells.

Heat maps showing A-to-G DNA on-target (blue) and A-to-G DNA off-target (orange) editing efficiencies of nCas9 (Control), ABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G each co-expressed with HEK site 2, HEK site 3, or HEK site 4 gRNAs (n=4 independent replicates). Editing windows shown include the most highly edited adenines. Numbering at the bottom represents spacer position with 1 being the most PAM-distal location.

Supplementary Figure 5 Additional data showing transcriptome-wide off-target RNA editing activities and sequence logos of non-rAPOBEC1 CBEs in human HEK293T cells.

(a) Manhattan plots showing transcriptome-wide distribution of RNA edits induced by hA3A-BE3 (these are the same data shown as Jitter plots in Fig. 3b). Sequence logos derived using all RNA-edited cytosines (0–100]% or stratified RNA-edited cytosines with high (80–100]%, middle (50–80]%, or low (0–50]% editing efficiencies induced by hA3A-BE3 expressed with the RNF2 gRNA. n = total number of modified cytosines. (b) Histograms showing the total number of RNA C-to-U edits observed (y-axis) at different editing efficiencies (x-axis) with expression of hA3A-BE3, eA3A-BE3, hAID-BE3, or Target-AID co-expressed with the RNF2 gRNA. Data from triplicate experiments are shown (derived from the data shown as Jitter plots in Fig. 3b). Dashed line = median; solid line = mean. Rep. = Replicate. n = number of modified cytosines.

Supplementary Figure 6 Self-editing generates a diverse range of heterogeneously edited CBE and ABE transcript sequences in HEK293T and HepG2 cells.

(a) Plots showing C-to-U self-editing of the BE3-encoding RNA transcript observed with WT BE3 (with rAPOBEC1) expression in HEK293T cells (sorted for all GFP-positive cells) with two different gRNAs targeting sites in RNF2 and EMX1. Each dot represents an edited C and the color of the dot indicates the predicted type of mutation caused by a C-to-U edit at that position (Methods). n = total number of modified cytosines. The y-axis shows editing efficiencies for each C-to-U change and the x-axis represents the position of each C within the BE3 coding sequence (with the architecture of the editor shown schematically below but not displaying the NLS and linkers). Data were obtained by analyzing additional replicates from previously published RNA-seq experiments5. (b) Plots illustrating C-to-U self-editing observed with wild-type (WT) BE3 (with rAPOBEC1), SECURE-BE3 (R33A) and SECURE-BE3 (R33A/K34A) in HEK293T and HepG2 cells sorted for top 5% GFP signal with co-expression of the RNF2 gRNA. Data are shown as described in a. Data were obtained by analyzing additional replicates from previously published RNA-seq experiments5. (c) Plots depicting C-to-U self-editing observed in HEK293T cells expressing hA3A-BE3, eA3A-BE3, hAID-BE3, and Target-AID (sorted for top 5% GFP signal). Data are shown as described in a and were obtained using additional replicates from the RNA-seq experiments shown in Fig. 3b. (d) Plots showing A-to-I self-editing induced by expression of ABEmax, miniABEmax, miniABEmax-K20A/R21A, and miniABEmax-V82G (sorted for all GFP-positive cells) each with a gRNA targeting HEK site 2, ABE site 16, or a non-targeting gRNA (NT) in HEK293T cells. Data are shown as described in a and were obtained using additional replicates from the RNA-seq experiments shown in Fig. 1c, d. n = total number of modified adenines.

Supplementary Figure 7 Assessment of gRNA editing induced by CBEs and ABEs in human cells.

(a) Plots showing C-to-U edits on gRNA observed with WT BE3 expression in HEK293T cells (sorted for all GFP-positive cells) and gRNAs targeting sites in RNF2 or EMX1. Data were obtained by analyzing previously published RNA-seq experiments5. (b) Plots illustrating C-to-U edits on gRNA observed with wild-type (WT) BE3, SECURE-BE3 (R33A) and SECURE-BE3 (R33A/K34A) in HEK293T and HepG2 cells sorted for top 5% GFP signal with co-expression of the RNF2 gRNA. Data were obtained by analyzing previously published RNA-seq experiments5. (c) Plots depicting C-to-U edits on gRNA observed in HEK293T cells expressing hA3A-BE3, eA3A-BE3, hAID-BE3, or Target-AID (sorted for top 5% GFP signal) with the RNF2 gRNA. Data were obtained from the RNA-seq experiments shown in Fig. 3b. (d) Plots showing A-to-I edits on gRNA induced by expression of ABEmax, miniABEmax, miniABEmax-K20A/R21A, or miniABEmax-V82G (sorted for all GFP-positive cells) each assessed with gRNAs targeting HEK site 2, ABE site 16, or a non-targeting gRNA (NT) in HEK293T cells. Data were obtained from the RNA-seq experiments shown in Fig. 1c, d. For all plots, each dot represents an edited C or A. n = total number of modified Cs or As; the y-axis shows editing efficiencies for each C-to-U or A-to-I modifications and the x-axis represents the position of each C or A within the gRNA sequence (spacer and scaffold).

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Grünewald, J., Zhou, R., Iyer, S. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 37, 1041–1048 (2019). https://doi.org/10.1038/s41587-019-0236-6

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