CRISPR/Cas9 Searches for a Protospacer Adjacent Motif by Lateral Diffusion

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2018 Dec 22

PMID 30573670

Citations 47

Authors

Viktorija Globyte

Seung Hwan Lee

Taegeun Bae

Jin-Soo Kim

Chirlmin Joo

Affiliations

Soon will be listed here.

Abstract

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.

Citing Articles

Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.

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Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.

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Deep learning based local feature classification to automatically identify single molecule fluorescence events.

Zhou S, Miao Y, Qiu H, Yao Y, Wang W, Chen C Commun Biol. 2024; 7(1):1404.

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CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

Soczek K, Cofsky J, Tuck O, Shi H, Doudna J bioRxiv. 2024; .

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