Investigating the metal-dependent function, allostery and inhibition of CRISPR-Cas9

研究 CRISPR-Cas9 的金属依赖性功能、变构和抑制

• 批准号: 10378667
• 负责人: Giulia Palermo
• 金额: $ 28.26万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10378667
• 关键词: Address; Affect; Allosteric Regulation; Applied Research; Basic Science; Behavior; Binding; Biological Sciences; Biology; Biophysics; CRISPR/Cas technology; Cancer Patient; Catalysis; Catalytic Domain; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complementary DNA; Complex; Computers; Computing Methodologies; DNA; DNA Binding; DNA Sequence; DNA Sequence Alteration; Data; Dependence; Development; Engineering; Enzymes; Event; Exhibits; Fostering; Free Energy; Gene Expression Regulation; Genetic Diseases; Genome; Grant; Guide RNA; Human Genetics; Investigation; Ionic Strengths; Ions; Kinetics; Knowledge; Malignant Neoplasms; Measurement; Mediating; Metabolism; Metals; Methods; Molecular Conformation; Nerve Degeneration; Nucleic Acid Binding; Nucleic Acids; Organism; Outcome; Proteins; Protocols documentation; Quantum Mechanics; Research; Ribonucleoproteins; Role; Safety; Sampling; Scientist; Series; Signal Transduction; Site; Specificity; System; T-Lymphocyte; Technology; Theoretical Studies; Variant; base; biophysical analysis; biophysical properties; clinical application; clinical efficacy; computer framework; computer studies; dalton; divalent metal; endonuclease; genome editing; graph theory; improved; improved functioning; inhibitor; innovation; molecular dynamics; molecular mechanics; nanosecond; network models; novel; nuclease; prevent; tool; transmission process; uptake; viral detection

Abstract CRISPR-Cas9 is the core of a transformative genome editing technology that is innovating life science with cutting-edge impact in basic and applied sciences. By enabling the correction of DNA mutations, this technology promises to treat a myriad of human genetic diseases, as shown for the first cancer patients treated with CRISPR-Cas9–modified T-cells. This technology is based on the endonuclease Cas9, which associates with guide RNAs to recognize and cleave complementary DNA sequences. Ceaseless development and engineering of CRISPR-Cas9 tools has opened novel intriguing hypotheses that grant in-depth investigations of the system. Here, the PI will implement unconventional multiscale approaches, combining a variety of state-of-the-art theoretical methods, to clarify the metal-dependent catalysis, the allostery in the selectivity mechanisms, as well as the inhibition of the system. We will pursue three specific aims, characterizing: (Aim 1) the DNA cleavage dependency on alternative divalent metal ions other than Mg2+ and the conformational effects associated with their binding; (Aim 2) the allosteric modulation witnessed in newly engineered Cas9 variants with enhanced specificity; (Aim 3) the inhibition mechanism by naturally occurring anti-CRISPR proteins to implement control over gene regulation. Toward these aims, we will leverage classical and enhanced sampling molecular dynamics (MD) simulations, high-level ab-initio MD (using the Car-Parrinello and Born- Oppenheimer approaches) and mixed quantum mechanics/molecular mechanics (QM/MM) approaches. Moreover, combination of ab-initio MD with graph theory will implement a synergistic approach capturing instantaneous sub-nanosecond signaling transfers. This will reveal how long-range allosteric effects impact the dynamics through evolving catalytic steps, elucidating the role of allostery in aiding catalysis. These multiscale approaches will offer a computational framework for the biophysical analysis of not only CRISPR-Cas9, but can also be extended to emerging CRISPR systems that are promising for genome editing and viral detection. Theoretical studies will be performed in close collaboration with experimental scientists, providing kinetic measurements and biophysical characterization, assisting in the interpretation of the experimental data and enabling testable predictions. Overall, this proposed research will expand the repertoire of mechanistic knowledge regarding the CRISPR-Cas9 function and lay the framework for novel engineering rationales toward improved genome editing.

摘要