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

Author

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  • Marius Rutkauskas

    (Universität Leipzig)

  • Inga Songailiene

    (Vilnius University)

  • Patrick Irmisch

    (Universität Leipzig)

  • Felix E. Kemmerich

    (Universität Leipzig)

  • Tomas Sinkunas

    (Vilnius University)

  • Virginijus Siksnys

    (Vilnius University)

  • Ralf Seidel

    (Universität Leipzig)

Abstract

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.

Suggested Citation

  • Marius Rutkauskas & Inga Songailiene & Patrick Irmisch & Felix E. Kemmerich & Tomas Sinkunas & Virginijus Siksnys & Ralf Seidel, 2022. "A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-35116-5
    DOI: 10.1038/s41467-022-35116-5
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    References listed on IDEAS

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    1. Samuel H. Sternberg & Benjamin LaFrance & Matias Kaplan & Jennifer A. Doudna, 2015. "Conformational control of DNA target cleavage by CRISPR–Cas9," Nature, Nature, vol. 527(7576), pages 110-113, November.
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    Cited by:

    1. Pierre Aldag & Marius Rutkauskas & Julene Madariaga-Marcos & Inga Songailiene & Tomas Sinkunas & Felix Kemmerich & Dominik Kauert & Virginijus Siksnys & Ralf Seidel, 2023. "Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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