Hybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules

Misha Klein, Behrouz Eslami Mosallam, D.W. Gonzalez Arroyo, Martin Depken

Research output: Contribution to journalArticleScientificpeer-review

57 Citations (Scopus)
82 Downloads (Pure)


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.
Original languageEnglish
Pages (from-to)1413-1423
JournalCell Reports
Issue number6
Publication statusPublished - 2018


  • OA-Fund TU Delft


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