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Mechanism, specificity, and design of CRISPR RNA-mediated gene regulation

CRISPR RNA介导的基因调控的机制、特异性和设计

基本信息

批准号：
10004678
负责人：
ILYA J FINKELSTEIN
金额：
$ 29.51万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004678
关键词：
Adaptive Immune System Address Archaea Archaeal RNA Bacteria Bacterial RNA Binding Biochemical Bioinformatics Biology Biophysical Process Biophysics Biotechnology Cells Chromatin Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Complex Crowding DNA DNA Binding DNA Sequence DNA-Binding Proteins Deoxyribonucleases Development Diffusion Dimensions Directed Molecular Evolution Electrostatics Elements Engineering Enzymes Eukaryotic Cell Family Future Gene Expression Gene Expression Regulation Gene Silencing Genes Genome Genome engineering Genomic Segment Genomics Goals Guide RNA Human Image Immune system Mediating Microscopy Modernization Molecular Mutation Nucleic Acids Nucleosomes Organism Play Process Proteins RNA Research Ribonucleases Role Scanning Specificity Structure System Techniques Testing Therapeutic Thymine Variant Virulence Virus adaptive immunity base clinical application design engineered nucleases experimental study fluorescence imaging genetic manipulation genome editing human pathogen imaging approach imaging platform improved insight interdisciplinary approach interest large scale data member nanoscale next generation novel nuclease off-target site precise genome editing predictive modeling programs protein profiling public health relevance single molecule tool

项目摘要

Project Summary Clustered regularly interspaced short palindromic repeats (CRISPRs) are a recently discovered RNA-based adaptive immune system that protects bacteria and archaea from foreign DNA. CRISPRs and the associated (Cas) proteins have been identified in 90% of archaea and 40% of bacteria, including many human pathogens. These immune systems also play a central role in controlling the horizontal transfer of virulence genes. The central step in CRISPR-Cas adaptive immunity is degradation of foreign DNA by a programmable RNA-guided nuclease. CRISPR-cas nucleases—enzymes that cleave a target DNA or RNA that is complementary to a guide CRISPR-RNA (crRNA)—have also been repurposed for precision gene regulation in many organisms. Despite the intense interest in these enzymes for both research and clinical applications, we still lack a complete understanding of their functions. Our long-term goal is to understand the mechanisms of CRISPR- mediated adaptive immunity. A related goal is to engineer improved CRISPR-associated enzymes for gene regulation and other biotechnological applications. The aims described in this proposal will mechanistically dissect a newly discovered family of RNA-guided nucleases. To achieve these aims, we pioneered high- throughput microscopy techniques that can image multiple enzymes and record their biochemical activities on tens of thousands of distinct DNA substrates. Using an interdisciplinary approach that integrates biophysics, bioinformatics, and micro-/nano-scale engineering, we will investigate how a CRISPR-associated RNA-guided nuclease recognizes and cleaves a target DNA. First, we will determine how the nuclease finds and cleaves a target DNA in the context of chromatin. Second, we will determine the biophysical mechanisms governing DNA binding and cleavage at off-target sites that resemble the on-target sequence. These off-target activities can cause unanticipated mutations that confound research experiments and limit therapeutic applications. Finally, we will engineer and profile novel high fidelity enzymes that reduce off-target activities and expand the genome engineering toolkit. CRISPR-mediated gene silencing and gene editing offers an exciting avenue for genetic manipulation of eukaryotic cells. We anticipate that our results will offer new insights in understanding the mechanisms of CRISPR-associated adaptive immunity and for using these enzymes for precision genome engineering in both scientific and future therapeutic settings.

项目总结

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.

DOI：
10.1038/s41587-020-0646-5
发表时间：
2021-01
期刊：
NATURE BIOTECHNOLOGY
影响因子：
46.9
作者：
Jones, Stephen K., Jr.;Hawkins, John A.;Johnson, Nicole V.;Jung, Cheulhee;Hu, Kuang;Rybarski, James R.;Chen, Janice S.;Doudna, Jennifer A.;Press, William H.;Finkelstein, Ilya J.
通讯作者：
Finkelstein, Ilya J.

CRISPR-Guided Programmable Self-Assembly of Artificial Virus-Like Nucleocapsids.

CRISPR指导的人造病毒样核素的可编程。

DOI：
10.1021/acs.nanolett.0c04640
发表时间：
2021-04-14
期刊：
NANO LETTERS
影响因子：
10.8
作者：
Calcines-Cruz, Carlos;Finkelstein, Ilya J.;Hernandez-Garcia, Armando
通讯作者：
Hernandez-Garcia, Armando

A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.

DOI：
10.1038/s41467-022-28994-2
发表时间：
2022-03-15
期刊：
Nature communications
影响因子：
16.6
作者：
Eslami-Mossallam B;Klein M;Smagt CVD;Sanden KVD;Jones SK Jr;Hawkins JA;Finkelstein IJ;Depken M
通讯作者：
Depken M

Massively parallel profiling of RNA-targeting CRISPR-Cas13d.