Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways

Joost Schimmel; Núria Muñoz-Subirana; H Kool; Robin van Schendel; Sven van der Vlies; Juliette Kamp; Femke de Vrij; Steven Kushner; Graeme C.M. Smith; SJ Boulton; Marcel Tijsterman

doi:10.1016/j.celrep.2023.112019

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways

Joost Schimmel, Núria Muñoz-Subirana, H Kool, Robin van Schendel, Sven van der Vlies, Juliette Kamp, Femke de Vrij, Steven Kushner, Graeme C.M. Smith, SJ Boulton, Marcel Tijsterman^*

^*Corresponding author for this work

Psychiatry

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Abstract

Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

Original language	English
Article number	112019
Journal	Cell Reports
Volume	42
Issue number	2
DOIs	https://doi.org/10.1016/j.celrep.2023.112019
Publication status	Published - 28 Feb 2023

Bibliographical note

Funding Information:
We thank Conny Brouwers (LUMC) for reagents and technical advice for the generation of GFP-Rosa-expressing cells. We thank Susan Kloet and Rolf Vossen from the Leiden Genome Technology Center for technical assistance. We thank Cristina Gontan Pardo (Erasmus MC) for generating the human GFP-AAVS1 iPSC line. S.J.B. is supported by the Francis Crick Institute (cc2098), a European Research Council (ERC) Advanced Investigator Grant (TelMetab), and Wellcome Trust Senior Investigator and Collaborative Grants; J.S. is supported by a Young Investigator Grant from the Dutch Cancer Society (KWF, 2020-1/12925); and M.T. is supported by grants from the Dutch Cancer Society (11251/2017-2) and the Holland Proton Therapy Centre (2019020-PROTON-DDR). J.S. N.M.-S. G.C.M.S. S.J.B. and M.T. developed the concept for the paper. J.S. N.M.-S. and H.K. generated mESC knockout cell lines, performed Cas9-targeting experiments, and generated NGS samples. R.v.S. wrote the custom SIQ program and analyzed sequence data. S.v.d.V. prepared samples for PacBio sequencing. J.A.K. performed the HDR experiment in human iPSCs under the supervision of F.d.V. and S.A.K. J.S. and N.M.-S. analyzed the data; J.S. N.M.-S. and M.T. interpreted the results and wrote the manuscript with input from all co-authors. S.J.B. is a co-founder, shareholder, and VP of Science Strategy at Artios Pharma, Ltd. Babraham Research Campus, UK. G.C.M.S. is chief scientific officer and shareholder of Artios Pharma, Ltd.

Funding Information:
We thank Conny Brouwers (LUMC) for reagents and technical advice for the generation of GFP-Rosa-expressing cells. We thank Susan Kloet and Rolf Vossen from the Leiden Genome Technology Center for technical assistance. We thank Cristina Gontan Pardo (Erasmus MC) for generating the human GFP-AAVS1 iPSC line. S.J.B. is supported by the Francis Crick Institute ( cc2098 ), a European Research Council ( ERC ) Advanced Investigator Grant ( TelMetab ), and Wellcome Trust Senior Investigator and Collaborative Grants; J.S. is supported by a Young Investigator Grant from the Dutch Cancer Society ( KWF , 2020-1/12925 ); and M.T. is supported by grants from the Dutch Cancer Society ( 11251/2017-2 ) and the Holland Proton Therapy Centre ( 2019020-PROTON-DDR ).

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© 2023 The Author(s)

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Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathwaysFinal published version, 2.96 MBLicence: CC BY

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Schimmel, J., Muñoz-Subirana, N., Kool, H., van Schendel, R., van der Vlies, S., Kamp, J., de Vrij, F., Kushner, S., Smith, G. C. M., Boulton, SJ., & Tijsterman, M. (2023). Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways. Cell Reports, 42(2), [112019]. https://doi.org/10.1016/j.celrep.2023.112019

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title = "Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways",

abstract = "Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.",

author = "Joost Schimmel and N{\'u}ria Mu{\~n}oz-Subirana and H Kool and {van Schendel}, Robin and {van der Vlies}, Sven and Juliette Kamp and {de Vrij}, Femke and Steven Kushner and Smith, {Graeme C.M.} and SJ Boulton and Marcel Tijsterman",

note = "Funding Information: We thank Conny Brouwers (LUMC) for reagents and technical advice for the generation of GFP-Rosa-expressing cells. We thank Susan Kloet and Rolf Vossen from the Leiden Genome Technology Center for technical assistance. We thank Cristina Gontan Pardo (Erasmus MC) for generating the human GFP-AAVS1 iPSC line. S.J.B. is supported by the Francis Crick Institute (cc2098), a European Research Council (ERC) Advanced Investigator Grant (TelMetab), and Wellcome Trust Senior Investigator and Collaborative Grants; J.S. is supported by a Young Investigator Grant from the Dutch Cancer Society (KWF, 2020-1/12925); and M.T. is supported by grants from the Dutch Cancer Society (11251/2017-2) and the Holland Proton Therapy Centre (2019020-PROTON-DDR). J.S. N.M.-S. G.C.M.S. S.J.B. and M.T. developed the concept for the paper. J.S. N.M.-S. and H.K. generated mESC knockout cell lines, performed Cas9-targeting experiments, and generated NGS samples. R.v.S. wrote the custom SIQ program and analyzed sequence data. S.v.d.V. prepared samples for PacBio sequencing. J.A.K. performed the HDR experiment in human iPSCs under the supervision of F.d.V. and S.A.K. J.S. and N.M.-S. analyzed the data; J.S. N.M.-S. and M.T. interpreted the results and wrote the manuscript with input from all co-authors. S.J.B. is a co-founder, shareholder, and VP of Science Strategy at Artios Pharma, Ltd. Babraham Research Campus, UK. G.C.M.S. is chief scientific officer and shareholder of Artios Pharma, Ltd. Funding Information: We thank Conny Brouwers (LUMC) for reagents and technical advice for the generation of GFP-Rosa-expressing cells. We thank Susan Kloet and Rolf Vossen from the Leiden Genome Technology Center for technical assistance. We thank Cristina Gontan Pardo (Erasmus MC) for generating the human GFP-AAVS1 iPSC line. S.J.B. is supported by the Francis Crick Institute ( cc2098 ), a European Research Council ( ERC ) Advanced Investigator Grant ( TelMetab ), and Wellcome Trust Senior Investigator and Collaborative Grants; J.S. is supported by a Young Investigator Grant from the Dutch Cancer Society ( KWF , 2020-1/12925 ); and M.T. is supported by grants from the Dutch Cancer Society ( 11251/2017-2 ) and the Holland Proton Therapy Centre ( 2019020-PROTON-DDR ). Publisher Copyright: {\textcopyright} 2023 The Author(s)",

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Schimmel, J, Muñoz-Subirana, N, Kool, H, van Schendel, R, van der Vlies, S, Kamp, J , de Vrij, F , Kushner, S, Smith, GCM, Boulton, SJ & Tijsterman, M 2023, 'Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways', Cell Reports, vol. 42, no. 2, 112019. https://doi.org/10.1016/j.celrep.2023.112019

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T1 - Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways

AU - Schimmel, Joost

AU - Muñoz-Subirana, Núria

AU - Kool, H

AU - van Schendel, Robin

AU - van der Vlies, Sven

AU - Kamp, Juliette

AU - de Vrij, Femke

AU - Kushner, Steven

AU - Smith, Graeme C.M.

AU - Boulton, SJ

AU - Tijsterman, Marcel

N1 - Funding Information: We thank Conny Brouwers (LUMC) for reagents and technical advice for the generation of GFP-Rosa-expressing cells. We thank Susan Kloet and Rolf Vossen from the Leiden Genome Technology Center for technical assistance. We thank Cristina Gontan Pardo (Erasmus MC) for generating the human GFP-AAVS1 iPSC line. S.J.B. is supported by the Francis Crick Institute (cc2098), a European Research Council (ERC) Advanced Investigator Grant (TelMetab), and Wellcome Trust Senior Investigator and Collaborative Grants; J.S. is supported by a Young Investigator Grant from the Dutch Cancer Society (KWF, 2020-1/12925); and M.T. is supported by grants from the Dutch Cancer Society (11251/2017-2) and the Holland Proton Therapy Centre (2019020-PROTON-DDR). J.S. N.M.-S. G.C.M.S. S.J.B. and M.T. developed the concept for the paper. J.S. N.M.-S. and H.K. generated mESC knockout cell lines, performed Cas9-targeting experiments, and generated NGS samples. R.v.S. wrote the custom SIQ program and analyzed sequence data. S.v.d.V. prepared samples for PacBio sequencing. J.A.K. performed the HDR experiment in human iPSCs under the supervision of F.d.V. and S.A.K. J.S. and N.M.-S. analyzed the data; J.S. N.M.-S. and M.T. interpreted the results and wrote the manuscript with input from all co-authors. S.J.B. is a co-founder, shareholder, and VP of Science Strategy at Artios Pharma, Ltd. Babraham Research Campus, UK. G.C.M.S. is chief scientific officer and shareholder of Artios Pharma, Ltd. Funding Information: We thank Conny Brouwers (LUMC) for reagents and technical advice for the generation of GFP-Rosa-expressing cells. We thank Susan Kloet and Rolf Vossen from the Leiden Genome Technology Center for technical assistance. We thank Cristina Gontan Pardo (Erasmus MC) for generating the human GFP-AAVS1 iPSC line. S.J.B. is supported by the Francis Crick Institute ( cc2098 ), a European Research Council ( ERC ) Advanced Investigator Grant ( TelMetab ), and Wellcome Trust Senior Investigator and Collaborative Grants; J.S. is supported by a Young Investigator Grant from the Dutch Cancer Society ( KWF , 2020-1/12925 ); and M.T. is supported by grants from the Dutch Cancer Society ( 11251/2017-2 ) and the Holland Proton Therapy Centre ( 2019020-PROTON-DDR ). Publisher Copyright: © 2023 The Author(s)

PY - 2023/2/28

Y1 - 2023/2/28

N2 - Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

AB - Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

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DO - 10.1016/j.celrep.2023.112019

M3 - Article

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VL - 42

JO - Cell Reports

JF - Cell Reports

IS - 2

M1 - 112019

ER -

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways

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