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Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes

Author

Listed:
  • Stephan Riesenberg

    (Max Planck Institute for Evolutionary Anthropology)

  • Philipp Kanis

    (Max Planck Institute for Evolutionary Anthropology)

  • Rosa Karlic

    (University of Zagreb)

  • Tomislav Maricic

    (Max Planck Institute for Evolutionary Anthropology)

Abstract

The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.

Suggested Citation

  • Stephan Riesenberg & Philipp Kanis & Rosa Karlic & Tomislav Maricic, 2025. "Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59947-0
    DOI: 10.1038/s41467-025-59947-0
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    References listed on IDEAS

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    1. Summer B. Thyme & Laila Akhmetova & Tessa G. Montague & Eivind Valen & Alexander F. Schier, 2016. "Internal guide RNA interactions interfere with Cas9-mediated cleavage," Nature Communications, Nature, vol. 7(1), pages 1-7, September.
    2. 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.
    3. Stephan Riesenberg & Tomislav Maricic, 2018. "Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells," Nature Communications, Nature, vol. 9(1), pages 1-9, December.
    4. Fatwa Adikusuma & Sandra Piltz & Mark A. Corbett & Michelle Turvey & Shaun R. McColl & Karla J. Helbig & Michael R. Beard & James Hughes & Richard T. Pomerantz & Paul Q. Thomas, 2018. "Large deletions induced by Cas9 cleavage," Nature, Nature, vol. 560(7717), pages 8-9, August.
    5. Peter C. DeWeirdt & Abby V. McGee & Fengyi Zheng & Ifunanya Nwolah & Mudra Hegde & John G. Doench, 2022. "Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    6. Michael Kosicki & Felicity Allen & Frances Steward & Kärt Tomberg & Yangyang Pan & Allan Bradley, 2022. "Cas9-induced large deletions and small indels are controlled in a convergent fashion," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    7. E. A. Moreb & M. D. Lynch, 2021. "Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
    8. Max W. Shen & Mandana Arbab & Jonathan Y. Hsu & Daniel Worstell & Sannie J. Culbertson & Olga Krabbe & Christopher A. Cassa & David R. Liu & David K. Gifford & Richard I. Sherwood, 2018. "Predictable and precise template-free CRISPR editing of pathogenic variants," Nature, Nature, vol. 563(7733), pages 646-651, November.
    9. Stephan Riesenberg & Nelly Helmbrecht & Philipp Kanis & Tomislav Maricic & Svante Pääbo, 2022. "Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    10. Daqi Wang & Chengdong Zhang & Bei Wang & Bin Li & Qiang Wang & Dong Liu & Hongyan Wang & Yan Zhou & Leming Shi & Feng Lan & Yongming Wang, 2019. "Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    11. Xi Xiang & Giulia I. Corsi & Christian Anthon & Kunli Qu & Xiaoguang Pan & Xue Liang & Peng Han & Zhanying Dong & Lijun Liu & Jiayan Zhong & Tao Ma & Jinbao Wang & Xiuqing Zhang & Hui Jiang & Fengping, 2021. "Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
    12. Grégoire Cullot & Julian Boutin & Jérôme Toutain & Florence Prat & Perrine Pennamen & Caroline Rooryck & Martin Teichmann & Emilie Rousseau & Isabelle Lamrissi-Garcia & Véronique Guyonnet-Duperat & Al, 2019. "CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    13. 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.
    14. 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.
    15. Anne Bothmer & Tanushree Phadke & Luis A. Barrera & Carrie M Margulies & Christina S. Lee & Frank Buquicchio & Sean Moss & Hayat S. Abdulkerim & William Selleck & Hariharan Jayaram & Vic E. Myer & Cec, 2017. "Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus," Nature Communications, Nature, vol. 8(1), pages 1-12, April.
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