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Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence

View ORCID ProfileFelicity Allen, View ORCID ProfileLuca Crepaldi, Clara Alsinet, Alexander J. Strong, Vitalii Kleshchevnikov, Pietro De Angeli, Petra Palenikova, Michael Kosicki, Andrew R. Bassett, Heather Harding, Yaron Galanty, Francisco Muñoz-Martínez, Emmanouil Metzakopian, Stephen P. Jackson, View ORCID ProfileLeopold Parts
doi: https://doi.org/10.1101/400341
Felicity Allen
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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  • ORCID record for Felicity Allen
  • For correspondence: leopold.parts@sanger.ac.uk fa9@sanger.ac.uk
Luca Crepaldi
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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  • ORCID record for Luca Crepaldi
Clara Alsinet
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Alexander J. Strong
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Vitalii Kleshchevnikov
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Pietro De Angeli
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Petra Palenikova
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Michael Kosicki
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Andrew R. Bassett
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Heather Harding
Cambridge Institute of Medical Research, University of Cambridge, Wellcome Trust MRC Building lab 6.36, Hills Road, Cambridge, CB2 0XY, United Kingdom
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Yaron Galanty
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom
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Francisco Muñoz-Martínez
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom
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Emmanouil Metzakopian
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom
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Stephen P. Jackson
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom
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Leopold Parts
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United KingdomDepartment of Computer Science, University of Tartu, J. Liivi 2, 51001, Tartu, Estonia
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  • For correspondence: leopold.parts@sanger.ac.uk fa9@sanger.ac.uk
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Abstract

The exact DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here, we present a systematic study of this link. We created a high throughput assay to directly measure the edits generated by over 40,000 guide RNAs, and applied it in a range of genetic backgrounds and for alternative CRISPR/Cas9 reagents. In total, we gathered data for over 1,000,000,000 mutational outcomes in synthetic constructs, which mirror those at endogenous loci. The majority of reproducible mutations are insertions of a single base, short deletions, or long microhomology-mediated deletions. gRNAs have a cell-line dependent preference for particular outcomes, especially favouring single base insertions and microhomology-mediated deletions. We uncover sequence determinants of the produced mutations at individual loci, and use these to derive a predictor of Cas9 editing outcomes with accuracy close to the theoretical maximum. This improved understanding of sequence repair allows better design of editing experiments, and may lead to future therapeutic applications.

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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-ND 4.0 International license.
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Posted August 25, 2018.
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Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence
Felicity Allen, Luca Crepaldi, Clara Alsinet, Alexander J. Strong, Vitalii Kleshchevnikov, Pietro De Angeli, Petra Palenikova, Michael Kosicki, Andrew R. Bassett, Heather Harding, Yaron Galanty, Francisco Muñoz-Martínez, Emmanouil Metzakopian, Stephen P. Jackson, Leopold Parts
bioRxiv 400341; doi: https://doi.org/10.1101/400341
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Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence
Felicity Allen, Luca Crepaldi, Clara Alsinet, Alexander J. Strong, Vitalii Kleshchevnikov, Pietro De Angeli, Petra Palenikova, Michael Kosicki, Andrew R. Bassett, Heather Harding, Yaron Galanty, Francisco Muñoz-Martínez, Emmanouil Metzakopian, Stephen P. Jackson, Leopold Parts
bioRxiv 400341; doi: https://doi.org/10.1101/400341

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