Constructing next-generation CRISPR–Cas tools from structural blueprints

Clustered regularly interspaced short palindromic repeats — CRISPR-associated protein (CRISPR–Cas) systems are a critical component of the bacterial adaptive immune response. Since the discovery that they can be reengineered as programmable RNA-guided nucleases, there has been significant interest in using these systems to perform diverse and precise genetic manipulations. Here, we outline recent advances in the mechanistic understanding of CRISPR–Cas9, how these findings have been leveraged in the rational redesign of Cas9 variants with altered activities, and how these novel tools can be exploited for biotechnology and therapeutics. We also discuss the potential of the ubiquitous, yet often-overlooked, multisubunit CRISPR effector complexes for large-scale genomic deletions. Furthermore, we highlight how future structural studies will bolster these technologies. •Target binding and R-loop formation by Cas9 require conformational rearrangements.•Structural insights enable the development of efficient and high-fidelity CRISPR–Cas9.•Advent of multisubunit CRISPR–Cas effectors as powerful gene editors in bacterial and human cells.