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Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

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  • Juan A. Perez-Bermejo

    (Graphite Bio)

  • Oghene Efagene

    (Graphite Bio)

  • William M. Matern

    (Graphite Bio)

  • Jeffrey K. Holden

    (Graphite Bio)

  • Shaheen Kabir

    (Graphite Bio)

  • Glen M. Chew

    (Graphite Bio)

  • Gaia Andreoletti

    (Graphite Bio)

  • Eniola Catton

    (Graphite Bio)

  • Craig L. Ennis

    (Graphite Bio)

  • Angelica Garcia

    (Graphite Bio)

  • Trevor L. Gerstenberg

    (Graphite Bio)

  • Kaisle A. Hill

    (Graphite Bio)

  • Aayami Jain

    (Graphite Bio)

  • Kristina Krassovsky

    (Graphite Bio)

  • Cassandra D. Lalisan

    (Graphite Bio)

  • Daniel Lord

    (Graphite Bio)

  • B. Joy Quejarro

    (Graphite Bio)

  • Jade Sales-Lee

    (Graphite Bio)

  • Meet Shah

    (Graphite Bio)

  • Brian J. Silva

    (Graphite Bio)

  • Jason Skowronski

    (Graphite Bio)

  • Yuri G. Strukov

    (Graphite Bio)

  • Joshua Thomas

    (Graphite Bio)

  • Michael Veraz

    (Graphite Bio)

  • Twaritha Vijay

    (Graphite Bio)

  • Kirby A. Wallace

    (Graphite Bio)

  • Yue Yuan

    (Graphite Bio)

  • Jane L. Grogan

    (Graphite Bio)

  • Beeke Wienert

    (Graphite Bio)

  • Premanjali Lahiri

    (Graphite Bio)

  • Sebastian Treusch

    (Graphite Bio)

  • Daniel P. Dever

    (Graphite Bio)

  • Vanessa B. Soros

    (Graphite Bio)

  • James R. Partridge

    (Graphite Bio)

  • Kristen L. Seim

    (Graphite Bio)

Abstract

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Suggested Citation

  • Juan A. Perez-Bermejo & Oghene Efagene & William M. Matern & Jeffrey K. Holden & Shaheen Kabir & Glen M. Chew & Gaia Andreoletti & Eniola Catton & Craig L. Ennis & Angelica Garcia & Trevor L. Gerstenb, 2024. "Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-46816-5
    DOI: 10.1038/s41467-024-46816-5
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    1. Sandra Wimberger & Nina Akrap & Mike Firth & Johan Brengdahl & Susanna Engberg & Marie K. Schwinn & Michael R. Slater & Anders Lundin & Pei-Pei Hsieh & Songyuan Li & Silvia Cerboni & Jonathan Sumner &, 2023. "Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    2. Ron Baik & M. Kyle Cromer & Steve E. Glenn & Christopher A. Vakulskas & Kay O. Chmielewski & Amanda M. Dudek & William N. Feist & Julia Klermund & Suzette Shipp & Toni Cathomen & Daniel P. Dever & Mat, 2024. "Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    3. Patrick L. Collins & Caitlin Purman & Sofia I. Porter & Vincent Nganga & Ankita Saini & Katharina E. Hayer & Greer L. Gurewitz & Barry P. Sleckman & Jeffrey J. Bednarski & Craig H. Bassing & Eugene M., 2020. "DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner," Nature Communications, Nature, vol. 11(1), pages 1-9, December.
    4. Daniel P. Dever & Rasmus O. Bak & Andreas Reinisch & Joab Camarena & Gabriel Washington & Carmencita E. Nicolas & Mara Pavel-Dinu & Nivi Saxena & Alec B. Wilkens & Sruthi Mantri & Nobuko Uchida & Ayal, 2016. "CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells," Nature, Nature, vol. 539(7629), pages 384-389, November.
    5. 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.
    6. Mara Pavel-Dinu & Volker Wiebking & Beruh T. Dejene & Waracharee Srifa & Sruthi Mantri & Carmencita E. Nicolas & Ciaran Lee & Gang Bao & Eric J. Kildebeck & Niraj Punjya & Camille Sindhu & Matthew A. , 2019. "Gene correction for SCID-X1 in long-term hematopoietic stem cells," Nature Communications, Nature, vol. 10(1), pages 1-15, December.
    7. Jennifer A. Doudna, 2020. "The promise and challenge of therapeutic genome editing," Nature, Nature, vol. 578(7794), pages 229-236, February.
    8. Pedro A. Mateos-Gomez & Fade Gong & Nidhi Nair & Kyle M. Miller & Eros Lazzerini-Denchi & Agnel Sfeir, 2015. "Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination," Nature, Nature, vol. 518(7538), pages 254-257, February.
    9. Natalia Gomez-Ospina & Samantha G. Scharenberg & Nathalie Mostrel & Rasmus O. Bak & Sruthi Mantri & Rolen M. Quadros & Channabasavaiah B. Gurumurthy & Ciaran Lee & Gang Bao & Carlos J. Suarez & Shauka, 2019. "Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    10. Adam C. Wilkinson & Daniel P. Dever & Ron Baik & Joab Camarena & Ian Hsu & Carsten T. Charlesworth & Chika Morita & Hiromitsu Nakauchi & Matthew H. Porteus, 2021. "Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
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