Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors

Nature Biotechnology volume 38, pages 875–882 (2020)Cite this article

Subjects

Abstract

Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited1. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C > T efficiency up to 61.61% and simultaneous C > T and A > G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Base editing of STEMEs via fused cytidine and adenosine deaminases.
Fig. 2: STEME-NG performs saturated mutagenesis in rice protoplasts.
Fig. 3: Directed evolution by saturated mutagenesis generates herbicide-tolerant mutants.

Similar content being viewed by others

Data availability

The authors declare that all data supporting the findings of this study are available in the article and its supplementary figures and tables or are available from the corresponding author upon request. For sequence data, rice LOC_Os IDs LOC_Os01g55540 (OsAAT), LOC_Os05g22940 (OsACC), LOC_Os03g05730 (OsCDC48), LOC_Os09g26999 (OsDEP1), LOC_Os02g11010 (OsOD, OsEV) are available on the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/). The deep sequencing data have been deposited with the NCBI BioProject database under accession code numbers PRJNA590652 and PRJNA590653. Plasmids encoding STEME-1, STEME-2, STEME-3, STEME-4, STEME-NG, A3A-PBE-NG, PABE-7-NG, pH-STEME-1-esgRNA and pH-STEME-NG-esgRNA will be available from Addgene.

Code availability

The custom Python script to analyze types of mutational reads and amino acid substitutions is in the Supplementary Code file.

References

  1. Engqvist, M. K. M. & Rabe, K. S. Applications of protein engineering and directed evolution in plant research. Plant Physiol. 179, 907–917 (2019).

    Article  CAS  Google Scholar 

  2. Henikoff, S., Till, B. J. & Comai, L. TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol. 135, 630–636 (2004).

    Article  CAS  Google Scholar 

  3. Slade, A. J., Fuerstenberg, S. I., Loeffler, D., Steine, M. N. & Facciotti, D. A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat. Biotechnol. 23, 75–81 (2005).

    Article  CAS  Google Scholar 

  4. Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015).

    Article  CAS  Google Scholar 

  5. Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011).

    Article  CAS  Google Scholar 

  6. Garst, A. D. et al. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nat. Biotechnol. 35, 48–55 (2016).

    Article  Google Scholar 

  7. Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018).

    Article  CAS  Google Scholar 

  8. Bao, Z. et al. Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision. Nat. Biotechnol. 36, 505–508 (2018).

    Article  CAS  Google Scholar 

  9. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  10. Ran, Y., Liang, Z. & Gao, C. Current and future editing reagent delivery systems for plant genome editing. Sci. China Life Sci. 60, 490–505 (2017).

    Article  CAS  Google Scholar 

  11. Butt, H. et al. CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors. Genome Biol. 20, 73 (2019).

    Article  Google Scholar 

  12. Zhang, Y. & Qi, Y. CRISPR enables directed evolution in plants. Genome Biol. 20, 83 (2019).

    Article  Google Scholar 

  13. 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 

  14. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, 1248 (2016).

    Article  CAS  Google Scholar 

  15. Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).

    Article  CAS  Google Scholar 

  16. Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).

    Article  CAS  Google Scholar 

  17. 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 

  18. Li, Q. et al. CRISPR–Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development. Nat. Cell Biol. 20, 1315–1325 (2018).

    Article  CAS  Google Scholar 

  19. Mol, C. D. et al. Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell 82, 701–708 (1995).

    Article  CAS  Google Scholar 

  20. 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 

  21. 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).

    Article  CAS  Google Scholar 

  22. Li, Z., Xiong, X. & Li, J.-F. New cytosine base editor for plant genome editing. Sci. China Life Sci. 61, 1602–1603 (2018).

    Article  Google Scholar 

  23. Li, C. et al. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion. Genome Biol. 19, 59 (2018).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. 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 

  26. Powles, S. B. & Yu, Q. Evolution in action: plants resistant to herbicides. Annu. Rev. Plant Biol. 61, 317–347 (2010).

    Article  CAS  Google Scholar 

  27. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    CAS  PubMed  Google Scholar 

  28. Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).

    Article  CAS  Google Scholar 

  29. Grünewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).

    Article  Google Scholar 

  30. Zhang, H., Yang, Z., Shen, Y. & Tong, L. Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase. Science 299, 2064–2067 (2003).

    Article  CAS  Google Scholar 

  31. Xing, H. L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

    Article  Google Scholar 

  32. Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).

    Article  CAS  Google Scholar 

  33. Meng, X. et al. Construction of a genome-wide mutant library in rice using CRISPR/Cas9. Mol. Plant 10, 1238–1241 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Zhang, H., Tweel, B. & Tong, L. Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfop and diclofop. Proc. Natl Acad. Sci. USA 101, 5910–5915 (2004).

    Article  CAS  Google Scholar 

  36. Xiang, S., Callaghan, M. M., Watson, K. G. & Tong, L. A different mechanism for the inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase by tepraloxydim. Proc. Natl Acad. Sci. USA 106, 20723–20727 (2009).

    Article  CAS  Google Scholar 

  37. Yu, L. P., Kim, Y. S. & Tong, L. Mechanism for the inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase by pinoxaden. Proc. Natl Acad. Sci. USA 107, 22072–22077 (2010).

    Article  CAS  Google Scholar 

  38. Eswar, N. et al. Tools for comparative protein structure modeling and analysis. Nucleic Acids Res. 31, 3375–3380 (2003).

    Article  CAS  Google Scholar 

  39. Bae, S., Park, J. & Kim, J.-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Jin for technical support on bioinformatic analysis. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, grant no. XDA24000000), the National Natural Science Foundation of China (grant nos. 31788103 and 31900301) and the National Key Research and Development Program of China (grant no. 2016YFD0101804).

Author information

Author notes

  1. These authors contributed equally: Chao Li, Rui Zhang, Xiangbing Meng.

Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Chao Li, Rui Zhang, Sha Chen, Yuan Zong, Chunju Lu & Caixia Gao

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Chao Li, Sha Chen, Yuan Zong, Chunju Lu, Jin-Long Qiu, Yu-Hang Chen, Jiayang Li & Caixia Gao

  3. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Xiangbing Meng & Jiayang Li

  4. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Jin-Long Qiu

  5. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China

    Yu-Hang Chen

Authors
  1. Chao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiangbing Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sha Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuan Zong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chunju Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jin-Long Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yu-Hang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jiayang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Caixia Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C. Li, R.Z., X.M., J.L. and C.G. designed the project. C. Li, R.Z., S.C., Y.Z. and C. Lu performed most of the experiments. X.M. performed transformation. Y.-H.C. analyzed the three-dimensional structure of the CT domain. J.L. and C.G. supervised the project. C. Li, R.Z., J.-L.Q., Y.-H.C., J.L. and C.G. wrote the manuscript.

Corresponding authors

Correspondence to Jiayang Li or Caixia Gao.

Ethics declarations

Competing interests

The authors have submitted a patent application based on the results reported in this paper.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Fig. 1 Activities and product purity of STEME-1 to STEME-4 in rice protoplasts.

(a)-(f) are OsAAT, OsACC, OsCDC48, OsDEP1, OsEV, and OsOD targets, respectively. One of three independent experiments is shown.

Supplementary Fig. 2 The allelic outcomes and indel value of STEMEs in rice protoplasts.

(a) The allelic outcomes of rice sites edited by STEME-1 (n=1 shown) in rice protoplasts. The simultaneous C>T and A>G editing efficiency of STEME-1 was ranged from 0.68-15.37% vs. 0.69-15.50% while only count the A>G substitutions (including single A>G substitution and simultaneous C>T and A>G substitutions) (Fig. 1d–f). (b) Indel frequencies of STEMEs and Cas9 in rice protoplasts. Comparison of indel frequencies by the A3A-PBE, PABE-7, STEME-1, STEME-2, STEME-3, and STEME-4 constructs in rice protoplasts. Values and error bars indicate the mean ± s.e.m of three independent experiments.

Supplementary Fig. 3 An overview of NG PAM targets at different rice loci.

(a) Architectures of the A3A-PBE-NG, PABE7-NG and pCas9-NG. ecTadA7.10, evolved Escherichia coli TadA; aa, amino acid; XTEN, a 16-aa linker; NLS, nuclear localization signal; CaMV, cauliflower mosaic virus; Term, terminator. (b) Sequences of 16 NG PAM targets in four loci of the rice genome. The PAM sequences are shown in red. (c) Diagram of OsAAT, OsCDC48, OsDEP1 and OsODEV, and their NG PAM targets. Regions flanking about 80bp of the OsAAT, OsCDC48, OsDEP1 and OsODEV loci were selected to design 20-nt sgRNA sequences to reduce the possible influence of chromatin state on editing.

Supplementary Fig. 4 Activities and product purity of STEME-NG in rice protoplasts.

(a)-(p) are NG PAM targets in OsAAT, OsCDC48, OsDEP1, and OsODEV, respectively. One of three independent experiments is shown.

Supplementary Fig. 5 Base editing efficiencies by A3A-PBE-NG and PABE7-NG in rice protoplasts.

Both A3A-PBE-NG (a) and PABE7-NG (b) had broad capacities for editing the targets of NGA, NGT, NGC or NGG PAMs. An untreated protoplast sample served as control. Values and error bars indicate the mean ± s.e.m of three independent experiments.

Supplementary Fig. 6 Indel frequencies of A3A-PBE-NG, PABE7-NG and STEME-NG compared with Cas9-NG in rice protoplasts.

A3A-PBE, PABE-7, STEME-1 and Cas9 together with NGG PAM sgRNAs served as positive control. The resulting efficiencies were log scaled by the base number 10. An untreated protoplast sample served as a negative control. Values and error bars indicate the mean ± s.e.m of three independent experiments.

Supplementary Fig. 7 Saturated mutagenesis of a 168-bp region of OsACC by A3A-PBE-NG in rice protoplasts.

A3A-PBE-NG (a) and the untreated control (b) are shown. Max values were calculated when different targets converted the same cytosine and guanine. Values and error bars indicate the mean ± s.e.m of three independent experiments.

Supplementary Fig. 8 C>T and A>G simultaneous editing and indel frequencies of 20 OsACC targets in rice protoplasts.

(a) The simultaneous editing frequencies of C>T and A>G in 20 targets of OsACC by STEME-NG in rice protoplasts. (b) Indel frequencies of 20 OsACC targets by A3A-PBE-NG, STEME-NG and Cas9-NG in rice protoplasts. An untreated protoplast sample served as a negative control. Values and error bars indicate the mean ± s.e.m of three independent experiments.

Supplementary Fig. 9 Saturated substitutions of 56 amino acids in the OsACC CT domain by A3A-PBE-NG in rice protoplasts.

Frequencies of silent, missense, and nonsense mutations were collected.

Supplementary Fig. 10 An overview of sgRNAs for saturated substituting 400 amino acids in the OsACC CT domain.

(a) An overview of the saturated targets on OsACC CT domain for rice plants. (b) Architectures of the binary vectors pH-STEME-1-esgRNA and pH-STEME-NG-esgRNA. ecTadA7.10, evolved Escherichia coli TadA; aa, amino acid; XTEN, a 16-aa linker; NLS, nuclear localization signal; E9 Term, pea rbcS-E9 terminator; pd35S, cauliflower mosaic virus double 35S promoter; Hyg, hygromycin. Arrows indicate the primers for amplicon deep sequencing.

Supplementary Fig. 11 Base editing and indel frequencies by STEMEs in rice plants.

(a) Base editing efficiencies of STEMEs in each transformed group. (b) Indel efficiencies of STEMEs in each transformed group. An average 0.90% indel reads in these groups, and 31.47% of the indels conferred in-frame mutations (Supplementary Dataset 2). Wild-type plants were used as untreated controls.

Supplementary Fig. 12 Genotypes of the P1927F, W2125C, S1866F, and A1884P mutants.

(a) The P1927F and Q1926* changes were caused by C:G>T:A transitions. Asterisk represents the stop codon. (b) The A2123T and W2125C changes were caused by a C:G>T:A transition and a C:G>G:C transversion, respectively. (c) The S1866F change was caused by a C:G>T:A transition and a silent mutation of G1869 was the result of a C:G>T:A transition. (d) The A1884P change was caused by a C:G>G:C transversion (A1884P-Hetero1) or simutaniously C:G>G:C transversion and A:T>G:C transition (A1884P-Hetero2). The edited bases are shown in lower case. Red triangles indicate peaks of edited bases. The genotypes of these mutations with two double peaks were confirmed further by T-vector cloning.

Supplementary Fig. 13 Selecting and genotyping haloxyfop resistance seedlings.

(a) Schematic of the procedure for mutating the OsACC CT domain via STEMEs on medium supplemented with haloxyfop using Groups 6 and 20 of individual sgRNAs. (b-c) The percentage of sgRNA sequencing reads after selection on medium containing 0.108 mg l-1 haloxyfop for 4 weeks in Group 6 (b) and Group 20 (c). The sgRNA OsACC-sg33 covered P1927 in Group 6 accounts for 100%, OsACC-sg140 and OsACC-sg141 both covered W2125 in Group 20 account for 96.74%. For the mutational reads of target region, 63.53% mutated sequences involved P1927F in Group 6, 48.71% mutated sequences involved W2125C in Group 20 (Supplementary Dataset 3). (d) Herbicide resistance seedlings harboring P1927F and W2125C mutations, respectively, regenerated in haloxyfop-containing medium. One biological experiment was performed. Scale bar, 2 cm. (e) Genotypes of herbicide resistance seedlings from Group 6 and Group 20. The P1927F and Q1926* were caused by C:G>T:A transitions; the A2123T was caused by C:G>T:A transition or C:G>T:A and A:T>G:C simultaneously conversions; the W2125C changes were caused by a C:G>G:C transversion. The edited bases are shown in lower case. Asterisk represents the stop codon. Red triangles indicate peaks of edited bases. The genotypes of these mutations with two double peaks were confirmed further by T-vector cloning.

Supplementary Fig. 14 Sequencing of OsACC full-length genomic DNA of P1927F, W2125C, S1866F, and A1884P mutants.

Twenty-two paired primers were used to sequence OsACC genomic DNA. The base changes of each mutant in P1927F, W2125C, S1866F, and A1884P codons were indicated. No other mutational changes occurred except for these target sites.

Supplementary Fig. 15 Phenotypes and genotypes of two A2123T mutants.

(a) Phenotypes of two homozygous A2123T mutants. One biological experiment was performed. Scale bar, 2 cm. Red arrows indicate the died symptom of newly grown leaves. (b) Genotypes of A2123T mutants. The A2123T change was caused by a C:G>T:A transition. The edited bases are shown in lower case. Red triangles indicate peaks of edited bases.

Supplementary information

Supplementary Materials

Supplementary Figs. 1–15, Supplementary Tables 1–8, Supplementary Sequences 1–3 and Supplementary Code.

Reporting Summary

Supplementary Dataset 1

Details of the saturated mutagenesis of 56 amino acids of the OsACC CT domain in rice protoplasts.

Supplementary Dataset 2

Mutational reads of directed evolution of 400 amino acids of the OsACC CT domain in rice plants.

Supplementary Dataset 3

Mutational reads of using STEMEs for targeted mutagenesis under concurrent selection pressure.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, C., Zhang, R., Meng, X. et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat Biotechnol 38, 875–882 (2020). https://doi.org/10.1038/s41587-019-0393-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-019-0393-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research