Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system

Pierre Aldag, Marius Rutkauskas, Julene Madariaga-Marcos, Inga Songailiene, Tomas Sinkunas, Felix Kemmerich, Dominik Kauert, Virginijus Siksnys () and Ralf Seidel ()
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Pierre Aldag: Universität Leipzig
Marius Rutkauskas: Universität Leipzig
Julene Madariaga-Marcos: Universität Leipzig
Inga Songailiene: Vilnius University
Tomas Sinkunas: Vilnius University
Felix Kemmerich: Universität Leipzig
Dominik Kauert: Universität Leipzig
Virginijus Siksnys: Vilnius University
Ralf Seidel: Universität Leipzig

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

Date: 2023
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DOI: 10.1038/s41467-023-38790-1

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