Existing adenine and cytosine base editors induce only a single type of modification, limiting the range of DNA alterations that can be created. Here we describe a CRISPR–Cas9-based synchronous programmable adenine and cytosine editor (SPACE) that can concurrently introduce A-to-G and C-to-T substitutions with minimal RNA off-target edits. SPACE expands the range of possible DNA sequence alterations, broadening the research applications of CRISPR base editors.
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Plasmids encoding SPACE have been deposited at Addgene (nos. 140242–140245). All RNA-seq next-generation sequencing data generated for this study have been deposited in the Gene Expression Omnibus data repository (series GSE137411). All targeted amplicon sequencing data (DNA on- and off-target editing) have been deposited at the Sequence Read Archive (PRJNA609075).
The authors will make all previously unreported custom computer code used in this work available upon request.
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
Grünewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).
Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Kaplanis, J. et al. Exome-wide assessment of the functional impact and pathogenicity of multinucleotide mutations. Genome Res. 29, 1047–1056 (2019).
Besenbacher, S. et al. Multi-nucleotide de novo mutations in humans. PLoS Genet. 12, e1006315 (2016).
Wang, Q. et al. Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes. Preprint at bioRxiv https://doi.org/10.1101/573378 (2019).
McKenna, A. et al. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 353, aaf7907 (2016).
Chan, M. M. et al. Molecular recording of mammalian embryogenesis. Nature 570, 77–82 (2019).
Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).
Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).
Li, C. et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat. Biotechnol. https://doi.org/10.1038/s41587-019-0393-7 (2020).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
McKenna, A. et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).
Karczewski, K.J. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Preprint at bioRxiv https://doi.org/10.1101/531210 (2020) https://storage.googleapis.com/gnomad-public/release/2.1/mnv/gnomad_mnv_coding.tsv
Mathelier, A. et al. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 44, D110–D115 (2016).
Schep, A. N., Wu, B., Buenrostro, J. D. & Greenleaf, W. J. chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nat. Methods 14, 975–978 (2017).
Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).
Support for this work was provided by the National Institutes of Health (RM1 HG009490 to J.K.J. 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 and the Robert B. Colvin, M. D. Endowed Chair in Pathology. J.G. was funded by the Deutsche Forschungsgemeinschaft (German Research Foundation) – Projektnummer 416375182. L.M.L. was supported by a Boehringer Ingelheim Fonds MD fellowship. We thank M. K. Clement for technical advice, K. Petri and P. K. Cabeceiras for discussions and technical advice, and L. Paul-Pottenplackel for assistance with editing the manuscript.
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). M.J.A. has financial interests in Excelsior Genomics. The interests of J.K.J. and M.J.A. 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., R.Z. and J.K.J. are co-inventors on a patent application that has been filed by Partners Healthcare/Massachusetts General Hospital on engineered programmable multi-deaminase base editor architectures to enable concurrent editing of distinct bases on DNA.
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Extended Data Fig. 1 Architectures of miniABEmax-V82G, Target-AID, and SPACE, and A-to-G or C-to-T editing distributions of miniABEmax-V82G or Target-AID.
a, Schematic illustration of miniABEmax-V82G, Target-AID, and SPACE architectures. Orange boxes = bipartite NLS for miniABEmax-V82G and SPACE or NLS for Target-AID; TadA* = mutant TadA 7.10 with V82G mutation, light grey box = SH3–3xFLAG for Target-AID, CDA1 = pmCDA1 with R187W mutation, and yellow boxes = UGIs. b, c, Box and dot plots indicating the distributions of A-to-G (pink, b) and C-to-T (blue, c) edits across 28 pooled genomic sites with miniABEmax-V82G (b) and Target-AID (c) including the entire protospacer. In box plots, the box spans the interquartile range (IQR) (first to third quartiles), the horizontal line shows the median (second quartile), and the whiskers extend to ±(1.5 × IQR). Single dots represent individual replicates. Graph was made using the same data as shown in Fig. 1b (n = 4).
Extended Data Fig. 2 On-target C-to-R (A/G) and A-to-Y (C/T) editing of miniABEmax-V82G, Target-AID, and SPACE.
Box and dot plots showing on-target DNA C-to-R (A/G) and A-to-Y (C/T) editing frequencies of miniABEmax-V82G (pink), Target-AID (blue), and SPACE (green) with 28 gRNAs (n = 4). Box, horizontal line, and whiskers are defined as in Fig. 1c. Single dots represent all Cs or As across the entire protospacer for all four replicates at each genomic site. Data from the same experiment as shown in Fig. 1b. Please note that box plots are mostly contained within the horizontal line (median, second quartile) close to or at the value ‘0’.
Dot plots showing on-target DNA indel frequencies induced by nCas9-D10A (Control, black), miniABEmax-V82G (pink), Target-AID (blue), and SPACE (green) with 28 gRNAs (n = 4). Single dots represent individual replicates. Data from the same experiment as shown in Fig. 1b.
Composition of alleles with frequencies of 1% or higher that result from SPACE editing with 27 gRNAs (HEK site 2 data shown in Fig. 1d). Data are taken from the first replicate obtained for each gRNA from the on-target experiment shown in Fig. 1b. Numbering indicates the position in the protospacer with 1 being the most PAM-distal location.
Extended Data Fig. 5 On-target DNA editing of SPACE compared to coexpression of miniABEmax-V82G and Target-AID (ABE & CBE mix) at 28 genomic sites.
Heat maps showing on-target DNA A-to-G (pink) and C-to-T (blue) editing frequencies induced by nCas9 (Control), coexpression of miniABEmax-V82G and Target-AID (ABE & CBE mix) or SPACE with 28 gRNAs (n = 4 independent replicates). Editing windows shown represent the edited adenines and cytosines, not the entirety of the protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal location.
Extended Data Fig. 6 On-target C-to-T, A-to-G, and dual editing, and indel frequencies induced by coexpression of miniABEmax-V82G and Target-AID compared with SPACE.
a, Bar and dot plots showing mean sum of allele frequencies of all edited alleles with A-to-G only, C-to-T only, and concurrent A-to-G and C-to-T editing resulting from coexpression of miniABEmax-V82G and Target-AID (ABE & CBE mix, grey) or SPACE (green) with 28 gRNAs (n = 4). Single dots represent individual replicates. Error bars represent the standard deviation (SD). Data are from the same experiment as shown in Extended Data Fig. 5. b, Dot plots showing on-target DNA indel frequencies of nCas9 (Control, black), coexpression of miniABEmax-V82G and Target-AID (ABE & CBE mix, grey) and SPACE (green) with 28 gRNAs (n = 4). Single dots represent individual replicates. Data are from the same experiment as shown in Extended Data Fig. 5.
Extended Data Fig. 7 Additional data and analysis from DNA and RNA off-target experiments and SPACE-inducible codon and amino-acid modifications.
a, Heat maps showing the on-target DNA A-to-G (pink) and C-to-T (blue) editing frequencies of nCas9 (Control), ABEmax, miniABEmax-V82G, Target-AID, or SPACE with HEK site 2 and RNF2 site 1 gRNAs (n = 3 independent replicates) for the RNA-seq experiments shown in Fig. 2a. Editing windows shown represent the most highly edited adenines and cytosines, not the entire protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal location. b, Histograms showing the total number of RNA A-to-I or C-to-U edits observed (y-axis) with different editing efficiencies (x-axis) for ABEmax, miniABEmax-V82G, Target-AID, or SPACE, each tested with the HEK site 2 and RNF2 site 1 gRNAs. n = number of modified adenines and cytosines. Experiments were performed in triplicate (data are from the same experiments as shown in Fig. 2a). Dashed red line, median; solid red line, mean. c, Heat maps showing on-target DNA A-to-G (pink) and C-to-T (blue) editing efficiencies of nCas9 (Control), miniABEmax-V82G, Target-AID, or SPACE with HEK sites 2–4, EMX1 site1, and FANCF site 1 gRNAs (n = 4 independent replicates) for DNA off-target experiments shown in Fig. 2b. Editing windows shown represent the most highly edited adenines and cytosines, not the entire protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal location. d, Circos plot showing 60 unique codon changes (with respect to the start codon) that can be induced by dual editing of adenines and cytosines by SPACE (grey), 18 of which (blue) lead to unique SPACE-inducible amino-acid changes with respect to the original codon (also see Fig. 2c and Supplementary Table 7).
Bar plot showing computationally determined number of genes (y-axis) that could be targeted by SPACE to install 1–5 (or more) transcription factor binding sites (x-axis) in proximity of the transcription start site of coding genes in the human genome for 10 transcription factors. Sites were filtered to contain a preferential SPACE editing window (C3-C4-A5) and a canonical NGG PAM (Methods).
Supplementary Fig. 1, Tables 3, 5 and 7 and Note.
List of all base editor constructs and controls with nucleotide and amino-acid sequences.
DNA on-target amplicon sequencing data for coexpression of miniABEmax-V82G and Target-AID (ABE & CBE mix) or expression of SPACE with 28 different gRNAs (for data presented in Extended Data Fig. 5).
List of potential targets from the gnomAD database for either correction or generation of MNVs by SPACE.
gRNA sequences, primer sequences and amplicon sequences.
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Grünewald, J., Zhou, R., Lareau, C.A. et al. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0535-y