CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells

Abstract

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications1,2,3,4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli–derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.

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Fig. 1: Engineering of a C-to-G base editor.
Fig. 2: Additional characterization of CGBE1 on-target editing activities in HEK293T cells.
Fig. 3: Comparison of CGBE1 and miniCGBE1 on-target editing activities in HEK293T cells.

Data availability

Plasmids encoding CGBE1 (Addgene no. 140252) and miniCGBE1 (Addgene no. 140253), as well as other constructs used in this work are available on Addgene via https://www.addgene.org/Keith_Joung/. Targeted amplicon sequencing data (obtained from Illumina Basespace) have been deposited at the Sequence Read Archive: https://www.ncbi.nlm.nih.gov/sra/PRJNA622835. All other relevant data are available from the corresponding authors upon request.

Code availability

No custom code was used in this study that was central to its conclusions. Code to generate plots from CRISPResso output tables will be provided upon request.

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Acknowledgements

This work was supported by the National Institutes of Health (grant nos. RM1 HG009490 and R35 GM118158) and by the Defense Advanced Research Projects Agency Safe Genes Program (grant no. HR0011-17-2-0042). J.G. was funded by the Deutsche Forschungsgemeinschaft (the German Research Foundation)—Projektnummer 416375182. L.M.L. was supported by a Boehringer Ingelheim Fonds MD fellowship. J.K.J. is additionally supported by the Robert B. Colvin, M.D. Endowed Chair in Pathology and the Desmond and Ann Heathwood Massachusetts General Hospital Research Scholar Award. We thank B. Kleinstiver and R. Walton for providing a codon-optimized version of VRQR-SpCas9 (D10A). We thank M.K. Clement for technical advice, K. Petri, P. Cabeceiras, J. Angstman, J. Hsu, C. Lareau and M. Aryee for discussions and technical advice and L. Paul-Pottenplackel for help with editing the manuscript. J.K.J. dedicates this work to the memory of K.S. Joung with gratitude for his lifelong advice, support and inspiration.

Author information

Affiliations

Authors

Contributions

S.I. and S.P.G. contributed equally to this work and are cosecond authors. Wet laboratory experiments were performed by I.C.K., R.Z., B.R.M. and L.M.L., while S.P.G. and S.I. performed computational analysis of the data. I.C.K., R.Z., J.G. and J.K.J. conceived and designed the study. J.G. and J.K.J. supervised the work. I.C.K., R.Z., J.G. and J.K.J. wrote the initial manuscript draft and all authors contributed to the editing of the manuscript.

Corresponding authors

Correspondence to Julian Grünewald or J. Keith Joung.

Ethics declarations

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). J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.G., I.C.K., R.Z. and J.K.J. are coinventors on patent applications that have been filed by Partners Healthcare/Massachusetts General Hospital on engineered C-to-G transversion base editors and other related base editor technologies.

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Extended data

Extended Data Fig. 1 On-target activities of nCas9 controls, ABE variants and more CBE variants tested for C-to-G editing in HEK293T cells.

Bar plots showing the on-target DNA base editing frequencies induced by nCas9 negative controls, ABE and ABE variants and other CBE variants with seven gRNAs in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side. Arrowheads indicate cytosines showing C-to-G edits by CGBE1.

Extended Data Fig. 2 Indel frequencies of nCas9 controls, ABE variants and CBE variants tested for C-to-G editing in HEK293T cells.

a, b, Dot plots representing percentage of alleles that contain an insertion or deletion across the entire protospacer from experiments with various base editor architectures reported in (a) Extended Data Fig. 1 for nCas9 controls and ABE variants or (b) Fig. 1b and Extended Data Fig. 1 for CBE variants and CGBE1. Single dots represent individual replicates (n = 3 independent replicates).

Extended Data Fig. 3 On-target activities of nCas9 controls and CGBE1-related variants with 12 C6 gRNAs in HEK293T cells.

Bar plots showing the on-target DNA base editing frequencies of nCas9 controls and CGBE1-related variants using 12 gRNAs for sites with a C at position 6 (C6-sites) in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side.

Extended Data Fig. 4 On-target activities of nCas9 controls and CGBE1-related variants with six non-C6 gRNAs in HEK293T cells and indel frequencies across 18 targeted sites.

a, Bar plots showing the on-target DNA base editing frequencies of nCas9 controls and CGBE1-related variants using six gRNAs for sites with a C at position 4, 5, 7, or 8 (non-C6-sites) in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side. b, Dot plots representing percentage of alleles that contain an insertion or deletion across the entire protospacer from experiments with CGBE1-related variants reported in Fig. 2a, b and Extended Data Figs. 3 and 4a. Single dots represent individual replicates (n = 3 independent replicates).

Extended Data Fig. 5 Aggregated distribution of C-to-G editing frequencies across protospacer with CGBE1 and miniCGBE1 in HEK293T cells.

a, b, Dot and box plots representing the aggregate distribution of C-to-G (yellow) editing frequencies per nucleotide across the entire protospacer from experiments performed with CGBE1 (a) and miniCGBE1 (b) with all 48 tested gRNAs. Boxes span the interquartile range (IQR; 25th to 75th percentile), horizontal lines indicate the median (50th percentile), and whiskers extend to ± 1.5 × IQR. Data points in plots represent full range of values plotted. Single dots represent individual replicates. The graphs were derived from the data shown in Fig. 3a, b (n = 4 independent replicates per site), and Supplementary Fig. 3a (n = 3 independent replicates per site).

Extended Data Fig. 6 On-target DNA editing activities of NG and VRQR variants of CGBE1 and miniCGBE1 in HEK293T cells.

a, Bar plots showing the on-target DNA base editing frequencies induced by NG and VRQR variants of nCas9, CGBE1, and miniCGBE1 using six gRNAs that target AT-rich genomic loci with PAMs that are compatible with SpCas9-NG (NGT) and SpCas9-VRQR (NGAG) variants in HEK293T cells. Editing frequencies of four independent replicates (n = 4) at each base are displayed side-by-side. b, Dot plots representing percentage of alleles that contain an insertion or deletion across the entire protospacer from experiments with NG and VRQR variants of CGBE1 and miniCGBE1 reported in a. Single dots or triangles represent individual replicates (n = 4 independent replicates).

Extended Data Fig. 7 Comparing the editing activities of CGBEs and PEs in multiple human cell lines.

a, Schematic of prime editing (PE) used to install a C-to-G substitution. PE fusion protein consists of an SpCas9-H840A nickase fused to an engineered Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The prime editing guide RNA (pegRNA) consists of a standard targetable SpCas9 gRNA that also harbors a 3’ extension containing a primer binding site (PBS) and a reverse transcription template (RTT) that encodes the desired edit. The PE2 system encompasses the prime editor fusion protein and a pegRNA. The PE3 system additionally includes a nicking gRNA (ngRNA). b, Bar plots showing the on-target DNA prime editing frequencies induced by nCas9(H840A), PE2 and PE3 using a pegRNA that targets FANCF site 1 across four human cancer cell lines. Gray overlay bars at top represent deletions at each nucleotide. Editing frequencies of four independent replicates (n = 4) for HEK293T cells or three independent replicates (n = 3) for K562, U2OS, and HeLa cells at each base are displayed side-by-side. Numbering on the bottom indicates the position of the base with 1 being the first nucleotide 3’ of the pegRNA/Cas9-induced nick. Arrowheads indicate guanines that exhibit desired G-to-T prime edits. c, d, Bar and dot plots representing the average on-target DNA prime editing and indel frequencies of PE2 and PE3 targeting FANCF site 1 for G-to-T prime editing (c, data from the same experiment as b) and HEK site 3 for PE-induced CTT insertion (d) in 4 cell lines. Single dots represent individual replicates (n = 4 for HEK293T and n = 3 for K562, U2OS, and HeLa cells). Error bars represent standard deviation (s.d.). Measure of center for the error bars = mean. e, Bar and dot plots showing the average on-target DNA C-to-G base or prime editing frequencies induced by CGBE1, miniCGBE1, PE2 or PE3 on four genomic target loci. Single dots represent individual replicates (n = 4 for HEK293T and n = 3 for K562, U2OS, and HeLa cells). A two-tailed Student’s t-test with p-values adjusted for multiple testing was used to calculate the shown p-values (p = 0.043 for both). Error bars represent (s.d.). Measure of center for the error bars = mean. f, Bar and dot plots representing the average frequency of alleles with indels (%) induced by pegRNAs and nicking gRNAs used in the experiments shown above (and FANCF site 1 +21 ngRNA control, Supplementary Table 9) with wild-type SpCas9 in HEK293T. pegRNAs/ngRNAs designed by Anzalone et al. (left) and by us (right) are separated by the dashed line. Single dots represent individual replicates (n = 3 independent replicates). Error bars represent (s.d.). Measure of center for the error bars = mean. ND, not done.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5.

Reporting Summary

Supplementary Table 1

Nucleotide and amino acid sequences of all base and prime editor constructs and controls used in this study.

Supplementary Table 2

DNA on-target amplicon sequencing data from screening studies performed with seven gRNAs (for data presented in Fig. 1b, Extended Data Fig. 1 and Supplementary Fig. 1a).

Supplementary Table 3

DNA on-target amplicon sequencing data of CGBE1-related variants and controls with 18 gRNAs (for data presented in Fig. 2a,b and Extended Data Figs. 3 and 4a).

Supplementary Table 4

DNA on-target amplicon sequencing data of CGBE1 and miniCGBE1 with 25 gRNAs (for data presented in Fig. 3 and Supplementary Fig. 2a).

Supplementary Table 5

DNA on-target amplicon sequencing data of CGBE1 and miniCGBE1 with 23 non-C6 gRNAs (for data presented in Supplementary Fig. 3a).

Supplementary Table 6

DNA off-target amplicon sequencing data of CGBE1 and miniCGBE1 (for data presented in Supplementary Fig. 4a).

Supplementary Table 7

DNA on-target amplicon sequencing data from experiments with NG and VRQR variants of CGBE1 and miniCGBE1 with six gRNAs each (for data presented in Extended Data Fig. 6a).

Supplementary Table 8

DNA on-target amplicon sequencing data of CGBEs compared to PEs in four different cell lines (for data presented in Extended Data Fig. 7b–e and Supplementary Fig. 5a).

Supplementary Table 9

Sequences of gRNAs and pegRNAs, primers and amplicons used in this study.

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Kurt, I.C., Zhou, R., Iyer, S. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0609-x

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