Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

Klein, M.; Eslami Mossallam, B.; Gonzalez Arroyo, D.W.; Depken, S.M.

doi:10.1016/j.celrep.2018.01.045

Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

Title

Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

Author

Klein, M. (TU Delft BN/Martin Depken Lab; Kavli institute of nanoscience Delft)
Eslami Mossallam, B. (TU Delft BN/Martin Depken Lab; Kavli institute of nanoscience Delft)
Gonzalez Arroyo, D.W. (Student TU Delft; Kavli institute of nanoscience Delft)
Depken, S.M. (TU Delft BN/Martin Depken Lab; Kavli institute of nanoscience Delft)

Date

2018

Abstract

Due to their specificity, efficiency, and ease of programming, CRISPR-associated nucleases are popular tools for genome editing. On the genomic scale, these nucleases still show considerable off-target activity though, posing a serious obstacle to the development of therapies. Off targeting is often minimized by choosing especially high-specificity guide sequences, based on algorithms that codify empirically determined off-targeting rules. A lack of mechanistic understanding of these rules has so far necessitated their ad hoc implementation, likely contributing to the limited precision of present algorithms. To understand the targeting rules, we kinetically model the physics of guide-target hybrid formation. Using only four parameters, our model elucidates the kinetic origin of the experimentally observed off-targeting rules, thereby rationalizing the results from both binding and cleavage assays. We favorably compare our model to published data from CRISPR-Cas9, CRISPR-Cpf1, CRISPR-Cascade, as well as the human Argonaute 2 system.

Subject

OA-Fund TU Delft

To reference this document use:

http://resolver.tudelft.nl/uuid:9a820451-405a-429c-a34b-cf56fc1c1e50

DOI

https://doi.org/10.1016/j.celrep.2018.01.045

ISSN

2211-1247

Source

Cell Reports, 22 (6), 1413-1423

Part of collection

Institutional Repository