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TREX1 restricts CRISPR-Cas9 genome editing in human cells

View ORCID ProfileMehmet E. Karasu, View ORCID ProfileEléonore Toufektchan, View ORCID ProfileJohn Maciejowski, View ORCID ProfileJacob E. Corn
doi: https://doi.org/10.1101/2022.12.12.520063
Mehmet E. Karasu
1Department of Biology, ETH Zurich, Zurich 8053, Switzerland
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Eléonore Toufektchan
2Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
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John Maciejowski
2Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
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Jacob E. Corn
1Department of Biology, ETH Zurich, Zurich 8053, Switzerland
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  • For correspondence: jacob.corn@biol.ethz.ch
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Abstract

CRISPR-Cas mediated homology-directed repair (HDR) can flexibly introduce desired mutations at targeted sites in a genome. But achieving high HDR efficiencies is a major hurdle in many cellular contexts. Moreover, cells from patients with mutations in DNA repair factors can exhibit low CRISPR-Cas-mediated HDR, complicating genome editing as a potential treatment. We used genome-wide screening in Fanconi anemia (FA) patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas mediated HDR. Surprisingly, we found that a single exonuclease called TREX1 is an important determinant of HDR efficiency when single-stranded templates are used as a repair template. TREX1 expression acts as a biomarker for CRISPR-Cas mediated HDR, such that cell lines expressing high levels of TREX1 have poor HDR that can be rescued by TREX1 removal. CRISPR-Cas mediated HDR can also be rescued by using single-stranded DNA templates that are chemically protected in a manner consistent with TREX1’s exonucleolytic activity. Overall, our data provide a mechanistic explanation for why some cells are easier to edit than others and suggest a route to increase CRISPR-Cas mediated HDR in TREX-expressing context.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 13, 2022.
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TREX1 restricts CRISPR-Cas9 genome editing in human cells
Mehmet E. Karasu, Eléonore Toufektchan, John Maciejowski, Jacob E. Corn
bioRxiv 2022.12.12.520063; doi: https://doi.org/10.1101/2022.12.12.520063
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TREX1 restricts CRISPR-Cas9 genome editing in human cells
Mehmet E. Karasu, Eléonore Toufektchan, John Maciejowski, Jacob E. Corn
bioRxiv 2022.12.12.520063; doi: https://doi.org/10.1101/2022.12.12.520063

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