"X" marks the spot: Mining the gold in CasX for gene editing.

In this issue of Molecular Cell, Tsuchida et al. (2022) present a successful structure-guided effort in improving genome-editing efficiencies of CRISPR-CasX from Deltaproteobacteria (DpbCasX) and Planctomycetes (PlmCasX). Engineered variants that stabilize the active conformational state improved the catalytic efficiency by ∼10-20 fold in vitro and mean-editing efficiency by ∼2-3 fold in human cells.

Coupled catalytic states and the role of metal coordination in Cas9.

Controlling the activity of the CRISPR-Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active Acidothermus cellulolyticus Cas9 complex along the reaction path at 2.2-2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of A. cellulolyticus Cas9 and commonly used Streptococcus pyogenes Cas9 in cells.

Catalytically Enhanced Cas9 through Directed Protein Evolution.

Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.

Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity.

Despite being utilized widely in genome sciences, CRISPR-Cas9 remains limited in achieving high fidelity in cleaving DNA. A better understanding of the molecular basis of Cas9 holds the key to improve Cas9-based tools. We employed direct evolution and in vitro characterizations to explore structural parameters that impact the specificity of the thermophilic Cas9 from Acidothermus cellulolyticus (AceCas9). By identifying variants that are able to cleave mismatched protospacers within the seed region, we found a critical role of the phosphate lock residues in substrate specificity in a manner that depends on their sizes and charges. Removal of the negative charge from the phosphate lock residues significantly decreases sensitivity to the guide-DNA mismatches. An increase in size of the substituted residues further reduces the sensitivity to mismatches at the first position of the protospacer. Our findings identify the phosphate lock residues as an important site for tuning the specificity and catalytic efficiency of Cas9.