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Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors

An Author Correction to this article was published on 10 June 2019

This article has been updated

Abstract

Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4–5 nucleotides to up to ~8–9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.

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Fig. 1: Cas9 PAM-variant ABE orthologs mediate A•T-to-G•C conversion in human cells.
Fig. 2: CP-Cas9 variants are compatible with the CBE and ABE architectures and exhibit broadened editing windows.

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Data availability

Plasmids encoding modified PAM adenine base editors and circularly permuted cytosine and adenine base editors have been deposited to Addgene. High-throughput sequencing data are deposited in the NCBI Sequence Read Archive (PRJNA498804).

Code availability

Access to and usage for the custom Crispresso2 script can be found in Supplementary Note 1 (ref. 39). ClinVar analysis of pathogenic human SNPs targetable by the base editors described in this study was executed using a custom Matlab script described previously1,2.

Change history

  • 10 June 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 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).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  Google Scholar 

  4. Zeng, Y. et al. Correction of the Marfan syndrome pathogenic FBN1 mutation by base editing in human cells and heterozygous embryos. Mol. Ther. 26, 2631–2637 (2018).

  5. 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).

    Article  CAS  Google Scholar 

  6. Chadwick, A. C., Wang, X. & Musunuru, K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler. Thromb. Vasc. Biol. 37, 1741–1747 (2017).

    Article  CAS  Google Scholar 

  7. Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).

    Article  Google Scholar 

  8. 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).

    Article  Google Scholar 

  9. Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

    Article  CAS  Google Scholar 

  10. Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).

    Article  CAS  Google Scholar 

  11. Yan, F. et al. Highly efficient A·T to G·C base editing by Cas9n-guided tRNA adenosine deaminase in rice. Mol. Plant 11, 631–634 (2018).

    Article  CAS  Google Scholar 

  12. Kang, B. C. et al. Precision genome engineering through adenine base editing in plants. Nat. Plants 4, 427–431 (2018).

    Article  CAS  Google Scholar 

  13. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016).

    Article  CAS  Google Scholar 

  14. Landrum, M. J. et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 42, D980–D985 (2014).

    Article  CAS  Google Scholar 

  15. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article  CAS  Google Scholar 

  16. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).

    Article  CAS  Google Scholar 

  17. Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    Article  CAS  Google Scholar 

  18. Li, X. et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).

    Article  CAS  Google Scholar 

  19. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  Google Scholar 

  20. Hua, K. et al. Precise A·T to G·C base editing in the rice genome. Mol. Plant 11, 627–630 (2018).

    Article  CAS  Google Scholar 

  21. Hua, K., Tao, X. & Zhu, J. K. Expanding the base editing scope in rice by using Cas9 variants. Plant Biotechnol. J. 17, 499–504 (2019).

  22. Yang, L. et al. Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 9, 814–819 (2018).

    Article  Google Scholar 

  23. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  CAS  Google Scholar 

  24. Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  Google Scholar 

  25. Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    Article  CAS  Google Scholar 

  26. Nishimasu, H. et al. Engineered CRISPR–Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    Article  CAS  Google Scholar 

  27. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  Google Scholar 

  28. Gu, T. et al. Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase. Chem. Sci. 9, 3248–3253 (2018).

    Article  CAS  Google Scholar 

  29. Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR–Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).

    Article  CAS  Google Scholar 

  30. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).

    Article  CAS  Google Scholar 

  31. Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018).

    Article  CAS  Google Scholar 

  32. Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).

  33. Jiang, W. et al. BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity. Cell Res. 28, 855–861 (2018).

  34. Ryu, S. M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).

    Article  CAS  Google Scholar 

  35. Oakes, B. L. et al. Circular permutants as programmable scaffolds for genome modification. Cell 176, 254–267 (2018).

    Article  Google Scholar 

  36. Jiang, F. et al. Structures of a CRISPR–Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).

    Article  CAS  Google Scholar 

  37. Huai, C. et al. Structural insights into DNA cleavage activation of CRISPR–Cas9 system. Nat. Commun. 8, 1375 (2017).

    Article  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by US NIH (grant nos. U01 AI142756, RM1 HG009490, R01 EB022376 and R35 GM118062); the St. Jude Research Consortium; the Ono Pharma Foundation; DARPA (grant no. HR0011–17–2–0049); and the HHMI. T.P.H. and K.T.Z. were supported by the Harvard Chemical Biology Program NIH Training Grant (no. T32 GM095450). S.M.M. was supported by an NSF graduate fellowship. B.L.O. was supported by the Innovative Genomic Institute Entrepreneurial Fellowship Program. C.F. is supported by US NIH grants (nos. K99 GM118909 and R00 GM118909). D.F.S. was supported by US NIH (grant no. DP2 EB018658).

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Authors and Affiliations

Authors

Contributions

T.P.H. and K.T.Z. conducted the experiments, performed analyses and wrote the manuscript. S.M.M. and N.M.G. conducted the experiments and performed analyses. B.L.O., C.F. and D.F.S. provided materials and advice on the CP variants. D.R.L. supervised the research and wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

The authors declare competing financial interests. D.R.L. is a consultant and cofounder of Editas Medicine, Pairwise Plants and Beam Therapeutics, companies that use genome editing. The authors have filed patent applications on base editors with altered targeting properties. The authors declare no competing non-financial interests.

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Integrated supplementary information

Supplementary Fig. 1 Indel frequencies for ABE PAM variants at six NGA PAM sites, six NGCG PAM sites or three other PAM sites.

(a) Percent of all sequencing reads containing an indel following modification by VRQR-ABEmax, xABEmax, ABEmax, or NG-ABEmax at six genomic sites containing an NGA PAM in HEK293T cells. (b) Percent of all sequencing reads containing an indel following modification by VRQR-ABEmax, VRER-ABEmax, xABEmax, ABEmax, or NG-ABEmax at six genomic sites containing an NGCG PAM in HEK293T cells. (c) Percent of all sequencing reads containing an indel following modification by VRQR-ABEmax, xABEmax, ABEmax, or NG-ABEmax at three genomic sites (PAM: GAT, CGCC, TGCC) in HEK293T cells. Values and error bars reflect the mean±s.d. of three independent biological replicates performed by different researchers on different days.

Supplementary Fig. 2 Peak editing position and indel frequencies for SaABEmax and SaKKH-ABEmax at six NNGRRT or six NNHRRT PAM sites.

(a) Top: Percent of all sequencing reads containing an indel following modification by SaABEmax or SaKKH-ABEmax at six genomic sites containing an NNGRRT PAM. Bottom: Representative sample of the top two allelic outcomes at six genomic sites containing NNGRRT PAMs following modification with SaABEmax (n = 1 shown) in HEK293T cells. The top allelic outcome is the unmodified amplicon, followed by the most common editing outcome being a single A-to-G conversion (4 out of 6 sites) within a shifted window (protospacer positions 7–11) (b) Top: Percent of all sequencing reads containing an indel following modification by SaABEmax or SaKKH-ABEmax at six genomic sites containing an NNHRRT PAM (where H = A, C, or T). Bottom: Representative sample of the top two allelic outcomes at six genomic sites containing NNHRRT PAMs following modification with SaKKH-ABEmax (n = 1 shown) in HEK293T cells. The top allelic outcome is the unmodified amplicon, followed by the most common editing outcome being a single A-to-G conversion (5 out of 6 sites) within a shifted window (protospacer positions 7–11). Values and error bars reflect the mean ± s.d. of three independent biological replicates performed by different researchers on different days.

Supplementary Fig. 3 Indel frequencies for CP1300-CBEmax and CP1300-ABEmax at five genomic sites.

(a) Percent of all sequencing reads containing an indel following modification by CP-CBEmax variants compared to CBEmax at five genomic sites containing a variety of adenines and cytosines in HEK293T cells. (b) Percent of all sequencing reads containing an indel following modification by CP-ABEmax variants compared to ABEmax at five genomic sites containing a variety of adenines and cytosines in HEK293T cells. Values and error bars reflect the mean ± s.d. of three independent biological replicates performed by different researchers on different days.

Supplementary Fig. 4 Base-editing frequency for CP1300-CBEmax and CP1300-ABEmax at five genomic sites containing adenines and cytosines.

Base editing with (a) CP1300-CBEmax and (b) CP1300-ABEmax at five genomic sites containing a variety of adenines and cytosines in HEK293T cells. Nucleobase conversion is highly site dependent, with minimal activity at most of the five genomic sites tested. Values and error bars reflect the mean ± s.d. of three independent biological replicates performed by different researchers on different days.

Supplementary Fig. 5 Out-of-protospacer C·G-to-T·A conversion by CP1012-CBEmax, CP1028-CBEmax and CP1041-CBEmax variants.

Three of the five genomic sites treated with CP-CBEmax variants exhibited both nontarget strand editing and out-of-protospacer editing. Representative samples of three sites are shown, with the protospacer designated by the grey box, and out-of-protospacer C·G-to-T·A conversion highlighted in the red box. CP1012-CBEmax exhibited the most frequent out-of-protospacer editing, with CP1028-CBEmax and CP1041-CBEmax exhibiting this property on only one of the sites.

Supplementary Fig. 6 Predicted average minimum distance between original or new C termini and the ssDNA substrate for base editing.

(a) Crystal structure of the SpCas9:gRNA:DNA ternary complex with the ssDNA bubble partially resolved (PDB: 5F9R)36. Novel CP termini are represented as spheres (original N- and C- termini in dark grey). The minimal linear distance between the predicted position of the novel CP termini (and WT C-terminus) and the furthest resolved nucleobase in the ssDNA bubble (corresponding to protospacer position 12, counting the PAM as positions 21–23) is depicted. (b) Cryo-EM structure of the SpCas9:gRNA:DNA ternary complex with the ssDNA bubble fully resolved (PDB: 5Y36)38. Novel CP termini are represented as spheres (original N- and C- termini in dark grey). The minimal linear distance between the predicted position of the novel CP termini (and WT C-terminus) and a protospacer position typically targeted for base editing (corresponding to protospacer position 4) is depicted. (c) Average of the distances to two different target positions on the ssDNA substrate measured for the novel CP termini (or WT C-terminus) from (a) and (b), listed in ascending order.

Supplementary Fig. 7 Edited product distribution for CP-CBEmax-B variants at two genomic sites prone to non-C-to-T byproduct formation.

The product distribution among edited DNA sequencing reads (reads in which the target C is base edited) is shown for each CBEmax variant with no UGI (“CBEmax-B” variants) tested at the same two sites as in Fig. 2g. Subscripted numbers indicate protospacer positions, counting the first base of the PAM as position 21. Values and error bars reflect the mean ± s.d. of three biological replicates performed on different days at each site. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, by two tailed Student’s t-test.

Supplementary Fig. 8 ClinVar analysis of targetable human pathogenic SNPs with expanded editing window CP-CBEmax and CP-ABEmax variants.

Fraction of pathogenic T•A-to-C•G to SNPs in ClinVar17,18 that could, in principle, be corrected by (a) CBEmax with an editing window of positions 4–8 (left) versus the SNPs correctable by CP-CBEmax with an editing window of positions 4–14 (right). Fraction of G•C-to-A•T pathogenic SNPs in ClinVar that could, in principle, be corrected by (b) ABEmax with an editing window of positions 4–8 (left) versus the SNPs correctable by CP-ABEmax with an editing window of positions 4–14 (right).

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Huang, T.P., Zhao, K.T., Miller, S.M. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat Biotechnol 37, 626–631 (2019). https://doi.org/10.1038/s41587-019-0134-y

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