Abstract
The RNA-guided endonuclease system CRISPR-Cas9 has been extensively modified since its discovery, allowing its capabilities to extend far beyond double-stranded cleavage to high fidelity insertions, deletions and single base edits. Such innovations have been possible due to the modular architecture of CRISPR-Cas9 and the robustness of its component parts to modifications and the fusion of new functional elements. Here, we review the broad toolkit of CRISPR-Cas9-based systems now available for diverse genome-editing tasks. We provide an overview of their core molecular structure and mechanism and distil the design principles used to engineer their diverse functionalities. We end by looking beyond the biochemistry and toward the societal and ethical challenges that these CRISPR-Cas9 systems face if their transformative capabilities are to be deployed in a safe and acceptable manner.
Highlights
Defined originally as an array of DNA repeats in 19871, the exact function of the clustered regularly interspaced short palindromic repeats (CRISPR) remained a mystery until the further discovery of CRISPR-associated (Cas) proteins and RNA elements
We explore the development of modified Cas9-based CRISPR systems for genome editing tasks, and the main approaches used to engineer these functionalities
In this review we have shown how robust the CRISPR-Cas[9] system is to modifications and extension, allowing its functionality to be tailored for a broad array of genome editing tasks in virtually any organism (Table 1)
Summary
Defined originally as an array of DNA repeats in 19871, the exact function of the clustered regularly interspaced short palindromic repeats (CRISPR) remained a mystery until the further discovery of CRISPR-associated (Cas) proteins and RNA elements. The T1337R mutation was found to be a gain of function, contrasting with the loss of function mutations utilized by other domain mutagenesis studies This specific gain of function permitted Cas[9] recognition of a fourth PAM base which increased the stringency of binding and reduced off-target effects compared to wild-type SpCas[951]. This alteration increased editing efficiencies in human cells 3-fold as UDG activity was drastically reduced[72] Both these editors are only active on the strand containing the cytosine so to broaden the editors’ function dCas[9] was modified to create variant BE3 that acted as a nickase targeting the non-edited strand (Figure 6C). Moving forward it will be essential that more reliable off-target detection methods are developed, as well as revisiting historic results to verify their accuracy
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call