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

Journal

Nature communications

ISSN: 2041-1723

Titre abrégé: Nat Commun

Pays: England

ID NLM: 101528555

Informations de publication

Date de publication:
23 Mar 2024

Historique:

received: 07 08 2023

accepted: 06 03 2024

medline: 24 3 2024

pubmed: 24 3 2024

entrez: 24 3 2024

Statut: epublish

Résumé

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.

Identifiants

DOI: 10.1038/s41467-024-46816-5 PMID: 38521763

pubmed: 38521763

doi: 10.1038/s41467-024-46816-5

pii: 10.1038/s41467-024-46816-5

doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

Pagination

2625

Informations de copyright

Références

Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).

pubmed: 32051598 pmcid: 8992613 doi: 10.1038/s41586-020-1978-5

Porteus, M. H. A new class of medicines through DNA editing. N. Engl. J. Med. 380, 947–959 (2019).

pubmed: 30855744 doi: 10.1056/NEJMra1800729

Morgan, R. A., Gray, D., Lomova, A. & Kohn, D. B. Hematopoietic stem cell gene therapy: progress and lessons learned. Cell Stem Cell 21, 574–590 (2017).

pubmed: 29100011 pmcid: 6039108 doi: 10.1016/j.stem.2017.10.010

Kohn, L. A. & Kohn, D. B. Gene therapies for primary immune deficiencies. Front. Immunol. 12, 648951 (2021).

pubmed: 33717203 pmcid: 7946985 doi: 10.3389/fimmu.2021.648951

Ferrari, G., Thrasher, A. J. & Aiuti, A. Gene therapy using haematopoietic stem and progenitor cells. Nat. Rev. Genet. 22, 216–234 (2021).

pubmed: 33303992 doi: 10.1038/s41576-020-00298-5

Nambiar, T. S., Baudrier, L., Billon, P. & Ciccia, A. CRISPR-based genome editing through the lens of DNA repair. Mol. Cell 82, 348–388 (2022).

pubmed: 35063100 pmcid: 8887926 doi: 10.1016/j.molcel.2021.12.026

Ferrari, S. et al. Gene editing of hematopoietic stem cells: hopes and hurdles toward clinical translation. Front. Genome Ed. 3, 618378 (2021).

pubmed: 34713250 pmcid: 8525369 doi: 10.3389/fgeed.2021.618378

Yeh, C. D., Richardson, C. D. & Corn, J. E. Advances in genome editing through control of DNA repair pathways. Nat. Cell Biol. 21, 1468–1478 (2019).

pubmed: 31792376 doi: 10.1038/s41556-019-0425-z

Shin, J. J. et al. Controlled cycling and quiescence enables efficient HDR in engraftment-enriched adult hematopoietic stem and progenitor cells. Cell Rep. 32, 108093 (2020).

pubmed: 32877675 pmcid: 7487781 doi: 10.1016/j.celrep.2020.108093

Ferrari, S. et al. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell 29, 1428–1444.e9 (2022).

pubmed: 36206730 pmcid: 9550218 doi: 10.1016/j.stem.2022.09.001

Allen, D. et al. High-throughput imaging of CRISPR- and recombinant adeno-associated virus-induced DNA damage response in human hematopoietic stem and progenitor cells. CRISPR J. 5, 80–94 (2022).

pubmed: 35049367 pmcid: 8892977 doi: 10.1089/crispr.2021.0128

Shy, B. R. et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat. Biotechnol. 41, 521–531 (2023).

pubmed: 36008610 doi: 10.1038/s41587-022-01418-8

De Ravin, S. S. et al. Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells. Blood 137, 2598–2608 (2021).

pubmed: 33623984 pmcid: 8120141 doi: 10.1182/blood.2020008503

Riesenberg, S. & Maricic, T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat. Commun. 9, 2164 (2018).

pubmed: 29867139 pmcid: 5986859 doi: 10.1038/s41467-018-04609-7

Canny, M. D. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol. 36, 95–102 (2018).

pubmed: 29176614 doi: 10.1038/nbt.4021

Selvaraj, S. et al. High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01888-4 (2023).

doi: 10.1038/s41587-023-01888-4 pubmed: 37537500

Wimberger, S. et al. Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing. Nat. Commun. 14, 4761 (2023).

pubmed: 37580318 pmcid: 10425386 doi: 10.1038/s41467-023-40344-4

Baik, R. et al. Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells. Nat. Commun. 15, 111 (2024).

pubmed: 38169468 pmcid: 10762240 doi: 10.1038/s41467-023-43413-w

Panier, S. & Boulton, S. J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).

pubmed: 24326623 doi: 10.1038/nrm3719

Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).

pubmed: 27820943 pmcid: 5898607 doi: 10.1038/nature20134

Wilkinson, A. C. et al. Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice. Nat. Commun. 12, 686 (2021).

pubmed: 33514718 pmcid: 7846836 doi: 10.1038/s41467-021-20909-x

Hoban, M. D. et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 125, 2597–2604 (2015).

pubmed: 25733580 pmcid: 4408287 doi: 10.1182/blood-2014-12-615948

Cromer, M. K. et al. Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nat. Med. 27, 677–687 (2021).

pubmed: 33737751 pmcid: 8265212 doi: 10.1038/s41591-021-01284-y

Gomez-Ospina, N. et al. Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I. Nat. Commun. 10, 4045 (2019).

pubmed: 31492863 pmcid: 6731271 doi: 10.1038/s41467-019-11962-8

Pavel-Dinu, M. et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat. Commun. 10, 1634 (2019).

pubmed: 30967552 pmcid: 6456568 doi: 10.1038/s41467-019-09614-y

Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018).

pubmed: 30082871 pmcid: 6107069 doi: 10.1038/s41591-018-0137-0

Lattanzi, A. et al. Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease. Sci. Transl. Med. 13, eabf2444 (2021).

pubmed: 34135108 pmcid: 8862191 doi: 10.1126/scitranslmed.abf2444

Mateos-Gomez, P. A. et al. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257 (2015).

pubmed: 25642960 pmcid: 4718306 doi: 10.1038/nature14157

Ramsden, D. A., Carvajal-Garcia, J. & Gupta, G. P. Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining. Nat. Rev. Mol. Cell Biol. 23, 125–140 (2022).

pubmed: 34522048 doi: 10.1038/s41580-021-00405-2

Xu, L. et al. Molecular dynamics of genome editing with CRISPR/Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease. Mol. Therapy Meth. Clin. Dev. 30, 317–331 (2023).

Collins, P. L. et al. DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner. Nat. Commun. 11, 3158 (2020).

pubmed: 32572033 pmcid: 7308414 doi: 10.1038/s41467-020-16926-x

Tatiossian, K. J. et al. Rational selection of CRISPR-Cas9 guide RNAs for homology-directed genome editing. Mol. Ther. 29, 1057–1069 (2021).

pubmed: 33160457 doi: 10.1016/j.ymthe.2020.10.006

Robert, F., Barbeau, M., Éthier, S., Dostie, J. & Pelletier, J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 7, 93 (2015).

pubmed: 26307031 pmcid: 4550049 doi: 10.1186/s13073-015-0215-6

Yue, X., Bai, C., Xie, D., Ma, T. & Zhou, P.-K. DNA-PKcs: A multi-faceted player in DNA damage response. Front. Genet. 11, 607428 (2020).

pubmed: 33424929 pmcid: 7786053 doi: 10.3389/fgene.2020.607428

Zenke, F. T. et al. Pharmacologic inhibitor of DNA-PK, M3814, potentiates radiotherapy and regresses human tumors in mouse models. Mol. Cancer Ther. 19, 1091–1101 (2020).

pubmed: 32220971 doi: 10.1158/1535-7163.MCT-19-0734

Berger, M. et al. BAY-8400: A novel potent and selective DNA-PK inhibitor which shows synergistic efficacy in combination with targeted alpha therapies. J. Med. Chem. 64, 12723–12737 (2021).

pubmed: 34428039 doi: 10.1021/acs.jmedchem.1c00762

Fok, J. H. L. et al. AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity. Nat. Commun. 10, 5065 (2019).

pubmed: 31699977 pmcid: 6838110 doi: 10.1038/s41467-019-12836-9

Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J. & Bao, G. COSMID: A web-based tool for identifying and validating CRISPR/Cas off-target sites. Mol. Ther. Nucleic Acids 3, e214 (2014).

pubmed: 25462530 pmcid: 4272406 doi: 10.1038/mtna.2014.64

Majeti, R., Park, C. Y. & Weissman, I. L. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 1, 635–645 (2007).

pubmed: 18371405 pmcid: 2292126 doi: 10.1016/j.stem.2007.10.001

Tomellini, E. et al. Integrin-α3 is a functional marker of ex vivo expanded human long-term hematopoietic stem cells. Cell Rep. 28, 1063–1073.e5 (2019).

pubmed: 31340144 doi: 10.1016/j.celrep.2019.06.084

Fares, I. et al. EPCR expression marks UM171-expanded CD34+ cord blood stem cells. Blood 129, 3344–3351 (2017).

pubmed: 28408459 doi: 10.1182/blood-2016-11-750729

Ferreira da Silva, J., Meyenberg, M. & Loizou, J. I. Tissue specificity of DNA repair: the CRISPR compass. Trends Genet. 37, 958–962 (2021).

pubmed: 34392967 doi: 10.1016/j.tig.2021.07.010

Fu, Y.-W. et al. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res. 49, 969–985 (2021).

pubmed: 33398341 pmcid: 7826255 doi: 10.1093/nar/gkaa1251

Hussmann, J. A. et al. Mapping the genetic landscape of DNA double-strand break repair. Cell 184, 5653–5669.e25 (2021).

pubmed: 34672952 pmcid: 9074467 doi: 10.1016/j.cell.2021.10.002

Roberts, B. et al. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol. Biol. Cell 28, 2854–2874 (2017).

pubmed: 28814507 pmcid: 5638588 doi: 10.1091/mbc.e17-03-0209

Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D., Biol. Crystallogr. 60, 2126–2132 (2004).

doi: 10.1107/S0907444904019158

Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. Sect. D., Biol. Crystallogr. 53, 240–255 (1997).

doi: 10.1107/S0907444996012255

Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

pubmed: 30809026 pmcid: 6533916 doi: 10.1038/s41587-019-0032-3

Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).

pubmed: 28459458 pmcid: 5924695 doi: 10.1038/nmeth.4278

Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

pubmed: 25513782 doi: 10.1038/nbt.3117

Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

pubmed: 16564017 doi: 10.1016/j.cell.2006.01.040

Edgar, R., Domrachev, M. & Lash, A. E. Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).

pubmed: 11752295 pmcid: 99122 doi: 10.1093/nar/30.1.207