A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity.

Journal Article (Journal Article)

CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here, we perform a meta-analysis of 39 published CRISPR-Cas9 data sets to understand better the role of secondary structures in inhibiting gRNA activity. We (1) identify two distinct inhibitory structures that can form, (2) measure the prevalence of these structures in existing gRNA library data sets, and (3) provide free energy cutoffs at which these structures become inhibitory. First, we show that hairpins that form within the targeting portion (spacer) of the gRNA, having a minimum free energy of <-5 kcal/mol, negatively impact gRNA activity. Second, we demonstrate that a longer hairpin can form between the spacer and the nexus portion of the gRNA scaffold. A duplex stability of this longer hairpin of <-15 kcal/mol negatively impacts gRNA activity. These cutoffs help to explain conflicting impacts of free energy values in different data sets, as well as provide a guideline for future gRNA designs.

Full Text

Duke Authors

Cited Authors

  • Moreb, EA; Lynch, MD

Published Date

  • February 2022

Published In

Volume / Issue

  • 5 / 1

Start / End Page

  • 146 - 154

PubMed ID

  • 35191752

Electronic International Standard Serial Number (EISSN)

  • 2573-1602

International Standard Serial Number (ISSN)

  • 2573-1599

Digital Object Identifier (DOI)

  • 10.1089/crispr.2021.0084


  • eng