Mitchell O. Roth

Area of research

Molecular Biology · Renewable Energy, Sustainability and the Environment

Research interest

Research topics from publications: Coupled catalytic states and the role of metal coordination in Cas9; Catalytically Enhanced Cas9 Through Directed Protein Evolution; Cas6 processes tight and relaxed repeat RNA via multiple mechanisms: A hypothesis; Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity; Solvent Accessibility of CRISPR-CAS9 Target DNA is Correlated with Substrate Specificity; A Catalytically Enhanced Type II-C Cas9 through Directed Protien Evolution; “X” marks the spot: Mining the gold in CasX for gene editing. Representative work: Abstract 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. Furtherm 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 Streptococcu

Looking for a research collaborator?

Search millions of scientists by field, institution, impact, and funding status — see their work, find their email, and reach out directly.