Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

Abstract

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

“…We believe a better understanding of how trL is naturally regulated will facilitate the development of controllable single guides. The recent creation of a theophyllineresponsive Cas9 RNP by the fusion of an aptamer to the sgRNA upper stem 61 underscores the potential for regulation to occur through the solvent-exposed 79 nt upper stem extension of trL. Furthermore, an elucidation of the structure and function of the Cas9:trL:dsDNA repressive complex could inform a new generation of Cas9-based transcriptional tools.…”

Engineering Cellular Signal Sensors based on CRISPR-sgRNA Reconstruction Approaches

“…The sgRNA that integrates the riboswitch properties allows the entire CRISPR system to acquire the ability to sense related ligands to regulate the "on-off" of the CRISPR system. Since the publication of our studies, there have been many studies attempting to modify sgRNAs in different ways to regulate the CRISPR system [40][41][42], depending on specific ligands. The interactions of ligands and the aptamers stabilize the structure of the aptamers, and this conformational change exposes the spacer of sgRNA blocked by the aptamer, which allows the sgRNA to restore the ability to guide the Cas protein to the targeted DNA sequence ( Figure 4A).…”

Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing

“…Kundert et al [64] rationally designed three ways to coordinate sgRNA conformation and activity: (1) grafting aptamers on different stem, (2) splitting sgRNA and recombining with aptamers, (3) inducing different base-pairing pattern. By screening the massive sgRNA library by in vitro assay, it is illustrated the last strategy is most successful in controlling CRISPR/Cas9 activity.…”