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Harnessing noncanonical crRNA for highly efficient genome editing

Author

Listed:
  • Guanhua Xun

    (University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign)

  • Zhixin Zhu

    (University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign)

  • Nilmani Singh

    (University of Illinois at Urbana-Champaign)

  • Jingxia Lu

    (University of Illinois at Urbana-Champaign)

  • Piyush K. Jain

    (University of Florida)

  • Huimin Zhao

    (University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign)

Abstract

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

Suggested Citation

  • Guanhua Xun & Zhixin Zhu & Nilmani Singh & Jingxia Lu & Piyush K. Jain & Huimin Zhao, 2024. "Harnessing noncanonical crRNA for highly efficient genome editing," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-48012-x
    DOI: 10.1038/s41467-024-48012-x
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    References listed on IDEAS

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    1. Winston X. Yan & Reza Mirzazadeh & Silvano Garnerone & David Scott & Martin W. Schneider & Tomasz Kallas & Joaquin Custodio & Erik Wernersson & Yinqing Li & Linyi Gao & Yana Federova & Bernd Zetsche &, 2017. "BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks," Nature Communications, Nature, vol. 8(1), pages 1-9, August.
    2. Nicholas S. McCarty & Alicia E. Graham & Lucie Studená & Rodrigo Ledesma-Amaro, 2020. "Multiplexed CRISPR technologies for gene editing and transcriptional regulation," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
    3. Liyang Zhang & John A. Zuris & Ramya Viswanathan & Jasmine N. Edelstein & Rolf Turk & Bernice Thommandru & H. Tomas Rube & Steve E. Glenn & Michael A. Collingwood & Nicole M. Bode & Sarah F. Beaudoin , 2021. "AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    4. 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.
    5. 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.
    6. Amanda R. Krysler & Christopher R. Cromwell & Tommy Tu & Juan Jovel & Basil P. Hubbard, 2022. "Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    7. Su Moon & Jeong Mi Lee & Jeong Gu Kang & Nan-Ee Lee & Dae-In Ha & Do Yon Kim & Sun Hee Kim & Kwangsun Yoo & Daesik Kim & Jeong-Heon Ko & Yong-Sam Kim, 2018. "Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    8. Andrew V. Anzalone & Peyton B. Randolph & Jessie R. Davis & Alexander A. Sousa & Luke W. Koblan & Jonathan M. Levy & Peter J. Chen & Christopher Wilson & Gregory A. Newby & Aditya Raguram & David R. L, 2019. "Search-and-replace genome editing without double-strand breaks or donor DNA," Nature, Nature, vol. 576(7785), pages 149-157, December.
    9. Changchang Xin & Jianhang Yin & Shaopeng Yuan & Liqiong Ou & Mengzhu Liu & Weiwei Zhang & Jiazhi Hu, 2022. "Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    10. Liyang Zhang & John A. Zuris & Ramya Viswanathan & Jasmine N. Edelstein & Rolf Turk & Bernice Thommandru & H. Tomas Rube & Steve E. Glenn & Michael A. Collingwood & Nicole M. Bode & Sarah F. Beaudoin , 2021. "Author Correction: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines," Nature Communications, Nature, vol. 12(1), pages 1-1, December.
    11. Rodrigo A. Gier & Krista A. Budinich & Niklaus H. Evitt & Zhendong Cao & Elizabeth S. Freilich & Qingzhou Chen & Jun Qi & Yemin Lan & Rahul M. Kohli & Junwei Shi, 2020. "High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening," Nature Communications, Nature, vol. 11(1), pages 1-9, December.
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