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A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex

Marius Rutkauskas, Inga Songailiene, Patrick Irmisch, Felix E. Kemmerich, Tomas Sinkunas, Virginijus Siksnys, Ralf Seidel
doi: https://doi.org/10.1101/2022.01.26.477710
Marius Rutkauskas
1Peter Debye Institute for Soft Matter Physics, Universität Leipzig, 04103 Leipzig, Germany
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Inga Songailiene
2Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekis ave. 7, Vilnius 10257, Lithuania
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Patrick Irmisch
1Peter Debye Institute for Soft Matter Physics, Universität Leipzig, 04103 Leipzig, Germany
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Felix E. Kemmerich
1Peter Debye Institute for Soft Matter Physics, Universität Leipzig, 04103 Leipzig, Germany
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Tomas Sinkunas
2Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekis ave. 7, Vilnius 10257, Lithuania
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Virginijus Siksnys
2Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekis ave. 7, Vilnius 10257, Lithuania
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Ralf Seidel
1Peter Debye Institute for Soft Matter Physics, Universität Leipzig, 04103 Leipzig, Germany
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  • For correspondence: ralf.seidel@physik.uni-leipzig.de
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SUMMARY

CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Contact Information: siksnys{at}ibt.lt

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted January 26, 2022.
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A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex
Marius Rutkauskas, Inga Songailiene, Patrick Irmisch, Felix E. Kemmerich, Tomas Sinkunas, Virginijus Siksnys, Ralf Seidel
bioRxiv 2022.01.26.477710; doi: https://doi.org/10.1101/2022.01.26.477710
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A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex
Marius Rutkauskas, Inga Songailiene, Patrick Irmisch, Felix E. Kemmerich, Tomas Sinkunas, Virginijus Siksnys, Ralf Seidel
bioRxiv 2022.01.26.477710; doi: https://doi.org/10.1101/2022.01.26.477710

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