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Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

Author

Listed:
  • Eman A. Ageely

    (Southern Illinois University)

  • Ramadevi Chilamkurthy

    (School of Medicine, Southern Illinois University)

  • Sunit Jana

    (McGill University)

  • Leonora Abdullahu

    (McGill University)

  • Daniel O’Reilly

    (McGill University
    RNA Therapeutics Institute, University of Massachusetts Medical School)

  • Philip J. Jensik

    (School of Medicine, Southern Illinois University)

  • Masad J. Damha

    (McGill University)

  • Keith T. Gagnon

    (Southern Illinois University
    School of Medicine, Southern Illinois University)

Abstract

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Suggested Citation

  • Eman A. Ageely & Ramadevi Chilamkurthy & Sunit Jana & Leonora Abdullahu & Daniel O’Reilly & Philip J. Jensik & Masad J. Damha & Keith T. Gagnon, 2021. "Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-26989-z
    DOI: 10.1038/s41467-021-26989-z
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    References listed on IDEAS

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    1. Carolin Anders & Ole Niewoehner & Alessia Duerst & Martin Jinek, 2014. "Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease," Nature, Nature, vol. 513(7519), pages 569-573, September.
    2. Hyo Min Park & Hui Liu & Joann Wu & Anthony Chong & Vanessa Mackley & Christof Fellmann & Anirudh Rao & Fuguo Jiang & Hunghao Chu & Niren Murthy & Kunwoo Lee, 2018. "Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo," Nature Communications, Nature, vol. 9(1), pages 1-12, December.
    3. De Dong & Kuan Ren & Xiaolin Qiu & Jianlin Zheng & Minghui Guo & Xiaoyu Guan & Hongnan Liu & Ningning Li & Bailing Zhang & Daijun Yang & Chuang Ma & Shuo Wang & Dan Wu & Yunfeng Ma & Shilong Fan & Jia, 2016. "The crystal structure of Cpf1 in complex with CRISPR RNA," Nature, Nature, vol. 532(7600), pages 522-526, April.
    4. Blake Wiedenheft & Samuel H. Sternberg & Jennifer A. Doudna, 2012. "RNA-guided genetic silencing systems in bacteria and archaea," Nature, Nature, vol. 482(7385), pages 331-338, February.
    5. Eunji Kim & Taeyoung Koo & Sung Wook Park & Daesik Kim & Kyoungmi Kim & Hee-Yeon Cho & Dong Woo Song & Kyu Jun Lee & Min Hee Jung & Seokjoong Kim & Jin Hyoung Kim & Jeong Hun Kim & Jin-Soo Kim, 2017. "In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni," Nature Communications, Nature, vol. 8(1), pages 1-12, April.
    6. Fernando Orden Rueda & Michal Bista & Matthew D. Newton & Anne U. Goeppert & M. Emanuela Cuomo & Euan Gordon & Felix Kröner & Jon A. Read & Jonathan D. Wrigley & David Rueda & Benjamin J. M. Taylor, 2017. "Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease," Nature Communications, Nature, vol. 8(1), pages 1-11, December.
    7. Christopher R. Cromwell & Keewon Sung & Jinho Park & Amanda R. Krysler & Juan Jovel & Seong Keun Kim & Basil P. Hubbard, 2018. "Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    8. Josiane E. Garneau & Marie-Ève Dupuis & Manuela Villion & Dennis A. Romero & Rodolphe Barrangou & Patrick Boyaval & Christophe Fremaux & Philippe Horvath & Alfonso H. Magadán & Sylvain Moineau, 2010. "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA," Nature, Nature, vol. 468(7320), pages 67-71, November.
    9. Alexis C. Komor & Yongjoo B. Kim & Michael S. Packer & John A. Zuris & David R. Liu, 2016. "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature, Nature, vol. 533(7603), pages 420-424, May.
    10. Long T. Nguyen & Brianna M. Smith & Piyush K. Jain, 2020. "Author Correction: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection," Nature Communications, Nature, vol. 11(1), pages 1-1, December.
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    Cited by:

    1. Jeong Moon & Changchun Liu, 2023. "Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA," Nature Communications, Nature, vol. 14(1), pages 1-11, December.

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