RT Journal Article
SR Electronic
T1 A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.26.477710
DO 10.1101/2022.01.26.477710
A1 Rutkauskas, Marius
A1 Songailiene, Inga
A1 Irmisch, Patrick
A1 Kemmerich, Felix E.
A1 Sinkunas, Tomas
A1 Siksnys, Virginijus
A1 Seidel, Ralf
YR 2022
UL http://biorxiv.org/content/early/2022/01/26/2022.01.26.477710.abstract
AB 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 StatementThe authors have declared no competing interest.