CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters

44Citations
Citations of this article
120Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

Background: Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment. Results: We present an approximate binding energy model for the Cas9-gRNA-DNA complex, which systematically combines the energy parameters obtained for RNA-RNA, DNA-DNA, and RNA-DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components. Conclusions: The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/.

Cite

CITATION STYLE

APA

Alkan, F., Wenzel, A., Anthon, C., Havgaard, J. H., & Gorodkin, J. (2018). CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters. Genome Biology, 19(1). https://doi.org/10.1186/s13059-018-1534-x

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free