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:
- 金额:$ 29.51万
- 依托单位:
- 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:
- 关键词:Adaptive Immune SystemAddressArchaeaArchaeal RNABacteriaBacterial RNABindingBiochemicalBioinformaticsBiologyBiophysical ProcessBiophysicsBiotechnologyCellsChromatinCleaved cellClustered Regularly Interspaced Short Palindromic RepeatsComplexCrowdingDNADNA BindingDNA SequenceDNA-Binding ProteinsDeoxyribonucleasesDevelopmentDiffusionDimensionsDirected Molecular EvolutionElectrostaticsElementsEngineeringEnzymesEukaryotic CellFamilyFutureGene ExpressionGene Expression RegulationGene SilencingGenesGenomeGenome engineeringGenomic SegmentGenomicsGoalsGuide RNAHumanImageImmune systemMediatingMicroscopyModernizationMolecularMutationNucleic AcidsNucleosomesOrganismPlayProcessProteinsRNAResearchRibonucleasesRoleScanningSpecificityStructureSystemTechniquesTestingTherapeuticThymineVariantVirulenceVirusadaptive immunitybaseclinical applicationdesignengineered nucleasesexperimental studyfluorescence imaginggenetic manipulationgenome editinghuman pathogenimaging approachimaging platformimprovedinsightinterdisciplinary approachinterestlarge scale datamembernanoscalenext generationnovelnucleaseoff-target siteprecise genome editingpredictive modelingprogramsprotein profilingpublic health relevancesingle moleculetool
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.
项目概要
成簇规则间隔短回文重复序列 (CRISPR) 是最近发现的一种基于 RNA 的
适应性免疫系统,保护细菌和古细菌免受外源 CRISPR 和相关 DNA 的侵害。
(Cas) 蛋白已在 90% 的古细菌和 40% 的细菌(包括许多人类病原体)中被发现。
这些免疫系统在控制毒力基因的水平转移方面也发挥着核心作用。
CRISPR-Cas适应性免疫的核心步骤是通过可编程RNA引导的外源DNA降解
CRISPR-cas 核酸酶——切割与 DNA 或 RNA 互补的目标 DNA 或 RNA 的酶。
引导 CRISPR-RNA (crRNA)——也已被重新用于许多生物体的精确基因调控。
尽管人们对这些酶的研究和临床应用产生了浓厚的兴趣,但我们仍然缺乏
我们的长期目标是了解 CRISPR 的机制。
介导的适应性免疫的一个相关目标是为基因设计改良的 CRISPR 相关酶。
本提案中描述的目标将机械地实现。
剖析新发现的 RNA 引导核酸酶家族 为了实现这些目标,我们开创了高-
通量显微镜技术可以对多种酶进行成像并记录它们的生化活性
使用整合生物物理学的跨学科方法,
生物信息学和微/纳米尺度工程,我们将研究 CRISPR 相关 RNA 引导如何
核酸酶识别并切割目标 DNA 首先,我们将确定核酸酶如何找到并切割目标 DNA。
其次,我们将确定控制 DNA 的生物物理机制。
在类似于目标序列的脱靶位点处进行结合和切割,这些脱靶活性可以。
导致意想不到的突变,从而扰乱研究实验并限制治疗应用。
我们将设计和分析新型高保真酶,以减少脱靶活性并扩展基因组
CRISPR 介导的基因沉默和基因编辑为遗传研究提供了一条令人兴奋的途径。
我们预计我们的结果将为理解真核细胞提供新的见解。
CRISPR 相关适应性免疫机制以及使用这些酶进行精准基因组研究
科学和未来治疗环境中的工程。
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:
- 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.
- DOI:10.1021/acs.nanolett.0c04640
- 发表时间:2021-04-14
- 期刊:
- 影响因子:10.8
- 作者:Calcines-Cruz, Carlos;Finkelstein, Ilya J.;Hernandez-Garcia, Armando
- 通讯作者:Hernandez-Garcia, Armando
Massively parallel profiling of RNA-targeting CRISPR-Cas13d.
- DOI:10.1038/s41467-024-44738-w
- 发表时间:2024-01-12
- 期刊:
- 影响因子:16.6
- 作者:Kuo, Hung-Che;Prupes, Joshua;Chou, Chia-Wei;Finkelstein, Ilya J.
- 通讯作者:Finkelstein, Ilya J.
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:
- 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
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ILYA J FINKELSTEIN的其他文献
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{{ truncateString('ILYA J FINKELSTEIN', 18)}}的其他基金
Turning a sequence barcode into a spectral barcode for single-cell analysis.
将序列条形码转换为光谱条形码以进行单细胞分析。
- 批准号:
9898410 - 财政年份:2019
- 资助金额:
$ 29.51万 - 项目类别:
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
- 批准号:
9365125 - 财政年份:2017
- 资助金额:
$ 29.51万 - 项目类别:
Mechanistic Characterization of the First Steps of Human DNA Break Repair
人类 DNA 断裂修复第一步的机制表征
- 批准号:
10001540 - 财政年份:2016
- 资助金额:
$ 29.51万 - 项目类别:
Mechanistic Characterization of the First Steps of Human DNA Break Repair
人类 DNA 断裂修复第一步的机制表征
- 批准号:
9752585 - 财政年份:2016
- 资助金额:
$ 29.51万 - 项目类别:
Mechanistic Characterization of the First Steps of Human DNA Break Repair
人类 DNA 断裂修复第一步的机制表征
- 批准号:
9323473 - 财政年份:2016
- 资助金额:
$ 29.51万 - 项目类别:
Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins
DNA 运动蛋白的染色质重塑和路障清除机制
- 批准号:
8616481 - 财政年份:2011
- 资助金额:
$ 29.51万 - 项目类别:
Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins
DNA 运动蛋白的染色质重塑和路障清除机制
- 批准号:
8090740 - 财政年份:2011
- 资助金额:
$ 29.51万 - 项目类别:
Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins
DNA 运动蛋白的染色质重塑和路障清除机制
- 批准号:
8636484 - 财政年份:2011
- 资助金额:
$ 29.51万 - 项目类别:
Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins
DNA 运动蛋白的染色质重塑和路障清除机制
- 批准号:
8829295 - 财政年份:2011
- 资助金额:
$ 29.51万 - 项目类别:
Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins
DNA 运动蛋白的染色质重塑和路障清除机制
- 批准号:
8251196 - 财政年份:2011
- 资助金额:
$ 29.51万 - 项目类别:
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