Anuska Das

Area of research

Molecular Biology · Genetics

Research interest

Research topics from publications: Coupled catalytic states and the role of metal coordination in Cas9; The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9; Structural principles of CRISPR-Cas enzymes used in nucleic acid detection; Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA; Directed evolution studies of a thermophilic Type II-C Cas9; Phosphate Lock Residues of Acidothermus cellulolyticus Cas9 Are Critical to Its Substrate Specificity; Structural and Biochemical Analysis of a Methylation Sensitive Cas9; Author response: Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA; Solvent Accessibility of CRISPR-CAS9 Target DNA is Correlated with Substrate Specificity. 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 Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to conf