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

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. Gerstenberg, Kaisle A. Hill, Aayami Jain, Kristina Krassovsky, Cassandra D. Lalisan, Daniel Lord, B. Joy Quejarro, Jade Sales-Lee, Meet Shah, Brian J. Silva, Jason Skowronski, Yuri G. Strukov, Joshua Thomas, Michael Veraz, Twaritha Vijay, Kirby A. Wallace, Yue Yuan, Jane L. Grogan, Beeke Wienert, Premanjali Lahiri, Sebastian Treusch, Daniel P. Dever, Vanessa B. Soros, James R. Partridge and Kristen L. Seim ()
Additional contact information
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

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-46816-5

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