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Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.

ABSTRACT: Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1-3 regions 'sense' nucleic acids, 'regulate' the HNH conformational transition, and ultimately 'lock' the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1-3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

SUBMITTER: Palermo G

PROVIDER: S-EPMC6292676 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.

Palermo Giulia G Chen Janice S JS Ricci Clarisse G CG Rivalta Ivan I Jinek Martin M Batista Victor S VS Doudna Jennifer A JA McCammon J Andrew JA

Quarterly reviews of biophysics 20180803

Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that th ...[more]

PMID: 30555184

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Project description:Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.

Dataset Information

Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.

Publications

Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.

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