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

Area of Science:

• Molecular Biology
• Bioinformatics

Background:

• CRISPR-Cas9 gene editing's efficacy is limited by off-target binding and cleavage.
• Guide RNA (gRNA) design significantly influences off-target activity.
• Computational prediction of off-targets is a viable alternative to experimental screening.

Purpose of the Study:

• To develop novel computational methods for assessing CRISPR-Cas9 off-target effects.
• To improve the selection of guide RNAs (gRNAs) for enhanced specificity and reduced off-target activity.
• To provide tools for accurate off-target evaluation in CRISPR-Cas9 applications.

Main Methods:

• An approximate binding energy model for the Cas9-gRNA-DNA complex was developed by combining energy parameters from RNA-RNA, DNA-DNA, and RNA-DNA duplexes.
• Two new methods, CRISPRoff and CRISPRspec, were introduced for off-target assessment and gRNA specificity measurement.
• The developed methods were benchmarked against existing state-of-the-art approaches.

Main Results:

• CRISPRoff and CRISPRspec demonstrated better agreement with experimental results compared to current methods.
• A significant inverse relationship was observed between on-target cleavage efficiency and system specificity.
• Binding energies were identified as key components influencing this inverse relationship.

Conclusions:

• The developed binding energy model offers insights into CRISPR-Cas9 off-targeting mechanisms.
• CRISPRoff and CRISPRspec enable more accurate off-target evaluation and gRNA selection for CRISPR-Cas9 genome editing.
• Webserver access is provided for CRISPRoff-based predictions and CRISPRspec-based evaluations: https://rth.dk/resources/crispr/