Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair.
Nat Commun
; 15(1): 2625, 2024 Mar 23.
Article
in En
| MEDLINE
| ID: mdl-38521763
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Recombinational DNA Repair
/
CRISPR-Cas Systems
Limits:
Humans
Language:
En
Journal:
Nat Commun
Journal subject:
BIOLOGIA
/
CIENCIA
Year:
2024
Document type:
Article
Affiliation country:
United States
Country of publication:
United kingdom