Abstract
CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.
Highlights
The three main targets for Cas endonucleases are double-stranded DNA, single-stranded DNA, and RNA, and we focus on the latter
Some Class 2 effectors, such as Cas13, naturally use only a single CRISPR RNA (crRNA) molecule. This natural gRNA contains a direct repeat (DR) stem-loop that mediates the interaction with the Cas protein and a spacer sequence that determines the target selectivity [62]
Experimental characterization of Cas systems has largely focused on cell applications, whereas in vitro characterization data, if available, are often buried in the supplementary information
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. DNA editing remains the most prominent area of CRISPR applications, bioengineers have increasingly turned their attention to more recently discovered Cas proteins capable of targeting and cleaving RNA instead of DNA [15,16]. Many in vitro Cas features that are critical for designing new applications, such as their mechanism of action, kinetics, and are not widely available for RNA-targeting CRISPR systems. Many in vitro Cas features that are critical for designing new applications, such as their mechanism of action, kinetics, and cleavage specificity, remain unknown or poorly documented.
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