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, Kristen L Seim
Author Information
Juan A Perez-Bermejo: Graphite Bio, South San Francisco, CA, USA. ORCID
Oghene Efagene: Graphite Bio, South San Francisco, CA, USA.
William M Matern: Graphite Bio, South San Francisco, CA, USA. ORCID
Jeffrey K Holden: Graphite Bio, South San Francisco, CA, USA.
Shaheen Kabir: Graphite Bio, South San Francisco, CA, USA.
Glen M Chew: Graphite Bio, South San Francisco, CA, USA. ORCID
Gaia Andreoletti: Graphite Bio, South San Francisco, CA, USA. ORCID
Eniola Catton: Graphite Bio, South San Francisco, CA, USA.
Craig L Ennis: Graphite Bio, South San Francisco, CA, USA.
Angelica Garcia: Graphite Bio, South San Francisco, CA, USA.
Trevor L Gerstenberg: Graphite Bio, South San Francisco, CA, USA.
Kaisle A Hill: Graphite Bio, South San Francisco, CA, USA.
Aayami Jain: Graphite Bio, South San Francisco, CA, USA.
Kristina Krassovsky: Graphite Bio, South San Francisco, CA, USA.
Cassandra D Lalisan: Graphite Bio, South San Francisco, CA, USA.
Daniel Lord: Graphite Bio, South San Francisco, CA, USA.
B Joy Quejarro: Graphite Bio, South San Francisco, CA, USA.
Jade Sales-Lee: Graphite Bio, South San Francisco, CA, USA.
Meet Shah: Graphite Bio, South San Francisco, CA, USA.
Brian J Silva: Graphite Bio, South San Francisco, CA, USA. ORCID
Jason Skowronski: Graphite Bio, South San Francisco, CA, USA.
Yuri G Strukov: Graphite Bio, South San Francisco, CA, USA.
Joshua Thomas: Graphite Bio, South San Francisco, CA, USA.
Michael Veraz: Graphite Bio, South San Francisco, CA, USA.
Twaritha Vijay: Graphite Bio, South San Francisco, CA, USA.
Kirby A Wallace: Graphite Bio, South San Francisco, CA, USA.
Yue Yuan: Graphite Bio, South San Francisco, CA, USA. ORCID
Jane L Grogan: Graphite Bio, South San Francisco, CA, USA. ORCID
Beeke Wienert: Graphite Bio, South San Francisco, CA, USA.
Premanjali Lahiri: Graphite Bio, South San Francisco, CA, USA.
Sebastian Treusch: Graphite Bio, South San Francisco, CA, USA.
Daniel P Dever: Graphite Bio, South San Francisco, CA, USA.
Vanessa B Soros: Graphite Bio, South San Francisco, CA, USA.
James R Partridge: Graphite Bio, South San Francisco, CA, USA.
Kristen L Seim: Graphite Bio, South San Francisco, CA, USA. scicomms@graphitebio.com. ORCID
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.
