SpringerLink
Log in

PAM-Less CRISPR-SpRY Genome Editing in Plants

  • Protocol
  • First Online:
Plant Genome Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2653))

Abstract

Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
EUR 44.95
Price includes VAT (Germany)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR 181.89
Price includes VAT (Germany)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
EUR 165.84
Price includes VAT (Germany)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info
Hardcover Book
EUR 235.39
Price includes VAT (Germany)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. **ek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci 93:1156–1160. https://doi.org/10.1073/pnas.93.3.1156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Boch J, Bonas U (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol 48:419–436. https://doi.org/10.1146/annurev-phyto-080508-081936

    Article  CAS  PubMed  Google Scholar 

  5. Miller JC, Tan S, Qiao G et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148. https://doi.org/10.1038/nbt.1755

    Article  CAS  PubMed  Google Scholar 

  6. Hefferin ML, Tomkinson AE (2005) Mechanism of DNA double-strand break repair by non-homologous end joining. DNA Repair 4:639–648. https://doi.org/10.1016/j.dnarep.2004.12.005

    Article  CAS  PubMed  Google Scholar 

  7. Sfeir A, Symington LS (2015) Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway? Trends Biochem Sci 40:701–714. https://doi.org/10.1016/j.tibs.2015.08.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liang F, Han M, Romanienko PJ et al (1998) Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci 95:5172–5177. https://doi.org/10.1073/pnas.95.9.5172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jeggo PA (1998) DNA breakage and repair. Adv Genet 38:185–218. https://doi.org/10.1016/s0065-2660(08)60144-3

  10. Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36:765–771. https://doi.org/10.1038/nbt.4192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Pickar-Oliver A, Gersbach CA (2019) The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Biol 20:490–507. https://doi.org/10.1038/s41580-019-0131-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Friedland AE, Baral R, Singhal P et al (2015) Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol 16:257. https://doi.org/10.1186/s13059-015-0817-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Walton RT, Christie KA, Whittaker MN et al (2020) Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368:290–296. https://doi.org/10.1126/science.aba8853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ren Q, Sretenovic S, Liu S et al (2021) PAM-less plant genome editing using a CRISPR–SpRY toolbox. Nat Plants 7:25–33. https://doi.org/10.1038/s41477-020-00827-4

    Article  CAS  PubMed  Google Scholar 

  15. Gaudelli NM, Komor AC, Rees HA et al (2017) Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature 551:464–471. https://doi.org/10.1038/nature24644

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nishida K, Arazoe T, Yachie N et al (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353(6305):aaf8729. https://doi.org/10.1126/science.aaf8729

  17. Komor AC, Kim YB, Packer MS et al (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424. https://doi.org/10.1038/nature17946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhao D, Li J, Li S et al (2021) Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39(1):35–40. https://doi.org/10.1038/s41587-020-0592-2

    Article  CAS  PubMed  Google Scholar 

  19. Chen L, Park JE, Paa P et al (2020) Precise and programmable C:G to G:C base editing in genomic DNA. bioRxiv:2020.07.21.213827. https://doi.org/10.1101/2020.07.21.213827

  20. Kurt IC, Zhou R, Iyer S et al (2021) CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol 39:41–46. https://doi.org/10.1038/s41587-020-0609-x

    Article  CAS  PubMed  Google Scholar 

  21. Kuscu C, Adli M (2016) CRISPR-Cas9-AID base editor is a powerful gain-of-function screening tool. Nat Methods 13:983–984. https://doi.org/10.1038/nmeth.4076

    Article  CAS  PubMed  Google Scholar 

  22. Wang X, Li J, Wang Y et al (2018) Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat Biotechnol 36:946–949. https://doi.org/10.1038/nbt.4198

    Article  CAS  PubMed  Google Scholar 

  23. Richter MF, Zhao KT, Eton E et al (2020) Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol 38(7):883–891. https://doi.org/10.1038/s41587-020-0453-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Grünewald J, Zhou R, Garcia SP et al (2019) Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569:433. https://doi.org/10.1038/s41586-019-1161-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Doman JL, Raguram A, Newby GA et al (2020) Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol 38:620–628. https://doi.org/10.1038/s41587-020-0414-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Xu Z, Kuang Y, Ren B et al (2021) SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition. Genome Biol 22:6. https://doi.org/10.1186/s13059-020-02231-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhang C, Wang Y, Wang F et al (2021) Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants. Mol Plant 14:191–194. https://doi.org/10.1016/j.molp.2020.12.016

    Article  CAS  PubMed  Google Scholar 

  28. Chen S, Liu Z, Lai L et al (2022) Efficient C-to-G base editing with improved target compatibility using engineered deaminase–nCas9 fusions. CRISPR J 5(3):389–396. https://doi.org/10.1089/crispr.2021.0124

  29. Sretenovic S, Liu S, Li G et al (2021) Exploring C-To-G base editing in rice, tomato, and poplar. Front Genome Ed 3:756766. https://doi.org/10.3389/fgeed.2021.756766

  30. Park J, Bae S, Kim J-S (2015) Cas-designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31(24):4014–4016. https://doi.org/10.1093/bioinformatics/btv537

    Article  CAS  PubMed  Google Scholar 

  31. Naito Y, Hino K, Bono H et al (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123. https://doi.org/10.1093/bioinformatics/btu743

    Article  CAS  PubMed  Google Scholar 

  32. Wu Y, He Y, Sretenovic S et al (2022) CRISPR-BETS: a base-editing design tool for generating stop codons. Plant Biotechnol J 20:499–510. https://doi.org/10.1111/pbi.13732

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the NSF Plant Genome Research Program (award no. IOS-2029889 and IOS-2132693) and the USDA-NIFA Biotechnology Risk Assessment Research Grants Program (award no. 2018-33522-28789) to Y.Q. It was also supported by the National Transgenic Major Project (award no. 2018ZX08020-003), the Technology Innovation and Application Development Program of Chongqing (award no. CSTC2021JSCX-CYLHX0001), and the Natural Science Foundation of Sichuan Province (award no. 2022NSFSC0143) to X.T. and Y.Z.

Author information

Authors and Affiliations

  1. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA

    Simon Sretenovic & Yi** Qi

  2. Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China

    Xu Tang, Qiurong Ren & Yong Zhang

  3. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA

    Yi** Qi

Authors
  1. Simon Sretenovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xu Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qiurong Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yi** Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yong Zhang or Yi** Qi .

Editor information

Editors and Affiliations

  1. Division of Plant Sciences, University of Missouri, Columbia, MO, USA

    Bing Yang

  2. Department of Crop Genetics, John Innes Centre, Norwich, UK

    Wendy Harwood

  3. Syngenta Crop Protection, LLC., Research Triangle Park, NC, USA

    Qiudeng Que

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sretenovic, S., Tang, X., Ren, Q., Zhang, Y., Qi, Y. (2023). PAM-Less CRISPR-SpRY Genome Editing in Plants. In: Yang, B., Harwood, W., Que, Q. (eds) Plant Genome Engineering. Methods in Molecular Biology, vol 2653. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3131-7_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3131-7_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3130-0

  • Online ISBN: 978-1-0716-3131-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation