IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v13y2022i1d10.1038_s41467-022-28884-7.html
   My bibliography  Save this article

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Author

Listed:
  • Annabel K. Sangree

    (Broad Institute of MIT and Harvard)

  • Audrey L. Griffith

    (Broad Institute of MIT and Harvard)

  • Zsofia M. Szegletes

    (Broad Institute of MIT and Harvard)

  • Priyanka Roy

    (Broad Institute of MIT and Harvard)

  • Peter C. DeWeirdt

    (Broad Institute of MIT and Harvard)

  • Mudra Hegde

    (Broad Institute of MIT and Harvard)

  • Abby V. McGee

    (Broad Institute of MIT and Harvard)

  • Ruth E. Hanna

    (Broad Institute of MIT and Harvard)

  • John G. Doench

    (Broad Institute of MIT and Harvard)

Abstract

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

Suggested Citation

  • Annabel K. Sangree & Audrey L. Griffith & Zsofia M. Szegletes & Priyanka Roy & Peter C. DeWeirdt & Mudra Hegde & Abby V. McGee & Ruth E. Hanna & John G. Doench, 2022. "Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28884-7
    DOI: 10.1038/s41467-022-28884-7
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-022-28884-7
    File Function: Abstract
    Download Restriction: no
    File URL: https://libkey.io/10.1038/s41467-022-28884-7?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    References listed on IDEAS

    as
    1. Johnny H. Hu & Shannon M. Miller & Maarten H. Geurts & Weixin Tang & Liwei Chen & Ning Sun & Christina M. Zeina & Xue Gao & Holly A. Rees & Zhi Lin & David R. Liu, 2018. "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity," Nature, Nature, vol. 556(7699), pages 57-63, April.
    2. Liwei Chen & Jung Eun Park & Peter Paa & Priscilla D. Rajakumar & Hong-Ting Prekop & Yi Ting Chew & Swathi N. Manivannan & Wei Leong Chew, 2021. "Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins," Nature Communications, Nature, vol. 12(1), pages 1-7, December.
    3. Josh Tycko & Michael Wainberg & Georgi K. Marinov & Oana Ursu & Gaelen T. Hess & Braeden K. Ego & Aradhana & Amy Li & Alisa Truong & Alexandro E. Trevino & Kaitlyn Spees & David Yao & Irene M. Kaplow , 2019. "Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    4. 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.
    5. Janice S. Chen & Yavuz S. Dagdas & Benjamin P. Kleinstiver & Moira M. Welch & Alexander A. Sousa & Lucas B. Harrington & Samuel H. Sternberg & J. Keith Joung & Ahmet Yildiz & Jennifer A. Doudna, 2017. "Enhanced proofreading governs CRISPR–Cas9 targeting accuracy," Nature, Nature, vol. 550(7676), pages 407-410, October.
    6. Gregory M. Findlay & Riza M. Daza & Beth Martin & Melissa D. Zhang & Anh P. Leith & Molly Gasperini & Joseph D. Janizek & Xingfan Huang & Lea M. Starita & Jay Shendure, 2018. "Accurate classification of BRCA1 variants with saturation genome editing," Nature, Nature, vol. 562(7726), pages 217-222, October.
    7. Benjamin P. Kleinstiver & Michelle S. Prew & Shengdar Q. Tsai & Ved V. Topkar & Nhu T. Nguyen & Zongli Zheng & Andrew P. W. Gonzales & Zhuyun Li & Randall T. Peterson & Jing-Ruey Joanna Yeh & Martin J, 2015. "Engineered CRISPR-Cas9 nucleases with altered PAM specificities," Nature, Nature, vol. 523(7561), pages 481-485, July.
    8. Richard W. Birkinshaw & Jia-nan Gong & Cindy S. Luo & Daisy Lio & Christine A. White & Mary Ann Anderson & Piers Blombery & Guillaume Lessene & Ian J. Majewski & Rachel Thijssen & Andrew W. Roberts & , 2019. "Structures of BCL-2 in complex with venetoclax reveal the molecular basis of resistance mutations," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    Full references (including those not matched with items on IDEAS)

    Citations

    Citations are extracted by the CitEc Project, subscribe to its RSS feed for this item.
    as


    Cited by:

    1. 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.

    Most related items

    These are the items that most often cite the same works as this one and are cited by the same works as this one.
    1. Péter István Kulcsár & András Tálas & Zoltán Ligeti & Eszter Tóth & Zsófia Rakvács & Zsuzsa Bartos & Sarah Laura Krausz & Ágnes Welker & Vanessza Laura Végi & Krisztina Huszár & Ervin Welker, 2023. "A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    2. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    3. Lin Zhao & Sabrina R. T. Koseki & Rachel A. Silverstein & Nadia Amrani & Christina Peng & Christian Kramme & Natasha Savic & Martin Pacesa & Tomás C. Rodríguez & Teodora Stan & Emma Tysinger & Lauren , 2023. "PAM-flexible genome editing with an engineered chimeric Cas9," Nature Communications, Nature, vol. 14(1), pages 1-8, December.
    4. Jian Wang & Yuxi Teng & Ruihua Zhang & Yifei Wu & Lei Lou & Yusong Zou & Michelle Li & Zhong-Ru Xie & Yajun Yan, 2021. "Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    5. Giulia I. Corsi & Kunli Qu & Ferhat Alkan & Xiaoguang Pan & Yonglun Luo & Jan Gorodkin, 2022. "CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    6. Angelo Miskalis & Shraddha Shirguppe & Jackson Winter & Gianna Elias & Devyani Swami & Ananthan Nambiar & Michelle Stilger & Wendy S. Woods & Nicholas Gosstola & Michael Gapinske & Alejandra Zeballos , 2024. "SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    7. Dawn G. L. Thean & Hoi Yee Chu & John H. C. Fong & Becky K. C. Chan & Peng Zhou & Cynthia C. S. Kwok & Yee Man Chan & Silvia Y. L. Mak & Gigi C. G. Choi & Joshua W. K. Ho & Zongli Zheng & Alan S. L. W, 2022. "Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    8. Jian Wang & Ke Wang & Zhe Deng & Zhiyu Zhong & Guo Sun & Qing Mei & Fuling Zhou & Zixin Deng & Yuhui Sun, 2024. "Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    9. Maarten H. Geurts & Shashank Gandhi & Matteo G. Boretto & Ninouk Akkerman & Lucca L. M. Derks & Gijs Son & Martina Celotti & Sarina Harshuk-Shabso & Flavia Peci & Harry Begthel & Delilah Hendriks & Pa, 2023. "One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    10. Luke Hoberecht & Pirunthan Perampalam & Aaron Lun & Jean-Philippe Fortin, 2022. "A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies," Nature Communications, Nature, vol. 13(1), pages 1-20, December.
    11. Fang Liang & Yu Zhang & Lin Li & Yexin Yang & Ji-Feng Fei & Yanmei Liu & Wei Qin, 2022. "SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    12. Zhaohui Zhong & Guanqing Liu & Zhongjie Tang & Shuyue Xiang & Liang Yang & Lan Huang & Yao He & Tingting Fan & Shishi Liu & Xuelian Zheng & Tao Zhang & Yiping Qi & Jian Huang & Yong Zhang, 2023. "Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    13. Marion Rosello & Malo Serafini & Luca Mignani & Dario Finazzi & Carine Giovannangeli & Marina C. Mione & Jean-Paul Concordet & Filippo Del Bene, 2022. "Disease modeling by efficient genome editing using a near PAM-less base editor in vivo," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    14. Zeyu Lu & Lingtian Zhang & Qing Mu & Junyang Liu & Yu Chen & Haoyuan Wang & Yanjun Zhang & Rui Su & Ruijun Wang & Zhiying Wang & Qi Lv & Zhihong Liu & Jiasen Liu & Yunhua Li & Yanhong Zhao, 2024. "Progress in Research and Prospects for Application of Precision Gene-Editing Technology Based on CRISPR–Cas9 in the Genetic Improvement of Sheep and Goats," Agriculture, MDPI, vol. 14(3), pages 1-17, March.
    15. Zsolt Bodai & Alena L. Bishop & Valentino M. Gantz & Alexis C. Komor, 2022. "Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    16. Jeremy Vicencio & Carlos Sánchez-Bolaños & Ismael Moreno-Sánchez & David Brena & Charles E. Vejnar & Dmytro Kukhtar & Miguel Ruiz-López & Mariona Cots-Ponjoan & Alejandro Rubio & Natalia Rodrigo Meler, 2022. "Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    17. Daniel C. Volke & Román A. Martino & Ekaterina Kozaeva & Andrea M. Smania & Pablo I. Nikel, 2022. "Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    18. Sundaram Acharya & Asgar Hussain Ansari & Prosad Kumar Das & Seiichi Hirano & Meghali Aich & Riya Rauthan & Sudipta Mahato & Savitri Maddileti & Sajal Sarkar & Manoj Kumar & Rhythm Phutela & Sneha Gul, 2024. "PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics," Nature Communications, Nature, vol. 15(1), pages 1-23, December.
    19. Qing Li & Jiansen Lu & Xidi Yin & Yunjian Chang & Chao Wang & Meng Yan & Li Feng & Yanbo Cheng & Yun Gao & Beiying Xu & Yao Zhang & Yingyi Wang & Guizhong Cui & Luang Xu & Yidi Sun & Rong Zeng & Yixue, 2023. "Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation," Nature Communications, Nature, vol. 14(1), pages 1-19, December.
    20. Grace N. Hibshman & Jack P. K. Bravo & Matthew M. Hooper & Tyler L. Dangerfield & Hongshan Zhang & Ilya J. Finkelstein & Kenneth A. Johnson & David W. Taylor, 2024. "Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9," Nature Communications, Nature, vol. 15(1), pages 1-15, December.

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28884-7. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    If CitEc recognized a bibliographic reference but did not link an item in RePEc to it, you can help with this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.