Spacer acquisition during the type III-A CRISPR-Cas immune response
III-A 型 CRISPR-Cas 免疫反应期间间隔区的获取
基本信息
- 批准号:10638980
- 负责人:
- 金额:$ 43.55万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-04-01 至 2027-02-28
- 项目状态:未结题
- 来源:
- 关键词:AddressArchaeaBacteriaBacteriophagesBase PairingBasic ScienceBindingBiotechnologyCellsCellular ImmunityClustered Regularly Interspaced Short Palindromic RepeatsComplexDNADNA SequenceDeoxyribonucleasesDevelopmentDiseaseEventGenerationsGenesGenetic DiseasesGenetic TranscriptionGenomeGenus staphylococcusGrowthGuide RNAHuman GeneticsImmune responseImmune systemImmunityInfectionIntegration Host FactorsInvadedInvestigationKnowledgeLibrariesLytic PhaseMediatingMemoryMicrococcal NucleaseMolecularMutagenesisNucleic AcidsPeriodicityPopulationProcessProductionRNARNA PhagesRibonucleasesSecond Messenger SystemsSingle-Stranded DNAStaphylococcus aureusSystemTestingTranscriptTranslational ResearchViralViral GenesViral GenomeVirusVirus Replicationadaptive immunityadenylatebacterial communityenzyme activityexperimental studygene therapyhuman pathogenmutantnucleasepreventresponsesample fixationsuccess
项目摘要
Project Summary
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against their viruses (phages).
The hallmark of the CRISPR-Cas immune response is the acquisition of a “memory” of infection in the form of
a short DNA sequence from the invading phage genome. This sequence, known as “spacer”, is integrated into
the CRISPR locus of the host and then transcribed and processed into a CRISPR RNA (crRNA) guide.
CRISPR-associated (Cas) effectors use the crRNA to recognize the nucleic acids of the invading phage
through base-pair complementarity and trigger different defense strategies. For Type III CRISPR systems,
commonly present in the human pathogen Staphylococcus aureus, target recognition leads to the dormancy of
the infected cell, an event that prevents viral replication and propagation. Here, we propose to investigate a
central, yet unanswered, question about this mechanism: if there are spacers that trigger a growth arrest in the
host, how are they maintained in the bacterial community after they are acquired? Our central hypothesis is
that the degradation of the phage DNA eventually eliminates the viral genome from the host, enabling growth
and the fixation of the spacer in the population. To investigate this, we will explore several aspects of the
Csm6-mediated response required for the defense mediated by type III CRISPR spacers that match late-
expressed viral genes. First, we will define whether dormant cells eventually die or are able to exit this state,
survive infection and continue growing. Second, we will determine whether spacers that match late-expressed
phage genes can provide a selective advantage to the cell that harbors them, even when they trigger host
dormancy. Third, we will determine if these spacers are actually acquired during infection. In all these
experiments we will test our central hypothesis by using mutant staphylococci lacking in the expression of
several nucleases to determine if they are required for the fixation of dormancy-triggering spacers. Finally, we
will use a transposon library of mutants to investigate, in an unbiased manner, the impact of host genes that
could be involved in the exit from dormancy.
Our proposed experiments, aimed at understanding how spacers from dormancy-inducing CRISPR systems
are fixed in the host population, will fill in a fundamental knowledge gap in our understanding of the hallmark
feature of CRISPR immunity: the generation of a memory of infection. In addition, by directly addressing a
fundamental mechanism of phage defense of staphylococci, our proposal can facilitate the success of phage
therapies for the treatment of staphylococcal disease. In a more indirect manner, the characterization of the
molecular mechanisms of type III CRISPR systems can lead to avenues to repurpose these immune systems
for gene editing, particularly for the development of gene therapies to treat genetic diseases.
项目摘要
CRISPR-Cas系统为细菌和古生菌提供了针对其病毒的适应性免疫。
CRISPR-Cas免疫应答的标志是以免疫应答的形式获得感染的“记忆”。
一段来自入侵噬菌体基因组的短DNA序列该序列被称为“间隔区”,被整合到
宿主的CRISPR基因座,然后转录并加工成CRISPR RNA(crRNA)向导。
CRISPR相关(Cas)效应物使用crRNA识别入侵噬菌体的核酸
通过碱基对的互补性,并引发不同的防御策略。对于III型CRISPR系统,
通常存在于人类病原体金黄色葡萄球菌中,目标识别导致
受感染的细胞,阻止病毒复制和繁殖的事件。在这里,我们建议调查一个
关于这一机制的一个核心但尚未回答的问题是:如果有间隔物在细胞中引发生长停滞,
宿主,它们在获得后如何在细菌群落中维持?我们的核心假设是
噬菌体DNA的降解最终消除了宿主中的病毒基因组,
以及间隔器在人群中的固定。为了研究这一点,我们将探讨几个方面的
由III型CRISPR间隔区介导的防御所需的Csm 6介导的应答,
表达病毒基因。首先,我们将定义休眠细胞最终是否死亡或能够退出这种状态,
在感染中存活并继续生长。其次,我们将确定是否匹配晚表达的
噬菌体基因可以为携带它们的细胞提供选择性优势,即使它们触发了宿主细胞,
休眠第三,我们将确定这些间隔区是否真的是在感染过程中获得的。在所有这些
实验中,我们将通过使用缺乏表达的突变葡萄球菌来测试我们的中心假设。
几种核酸酶,以确定它们是否是固定休眠触发间隔区所需的。最后我们
将使用转座子突变体库,以无偏见的方式调查宿主基因的影响,
可能参与了休眠的退出
我们提出的实验旨在了解诱导休眠的CRISPR系统中的间隔区如何
在宿主人群中是固定的,将填补我们对标志性疾病的理解中的一个基本知识空白,
CRISPR免疫的一个重要特征:产生感染记忆。此外,通过直接寻址
噬菌体防御葡萄球菌的基本机制,我们的建议可以促进噬菌体的成功
用于治疗葡萄球菌疾病的疗法。以一种更间接的方式,
III型CRISPR系统的分子机制可以导致重新利用这些免疫系统的途径
用于基因编辑,特别是用于开发治疗遗传疾病的基因疗法。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Luciano A Marraffini其他文献
Luciano A Marraffini的其他文献
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{{ truncateString('Luciano A Marraffini', 18)}}的其他基金
Generation of immunological memory by CRISPR-Cas systems
通过 CRISPR-Cas 系统生成免疫记忆
- 批准号:
9750114 - 财政年份:2017
- 资助金额:
$ 43.55万 - 项目类别:
Generation of immunological memory by CRISPR-Cas systems
通过 CRISPR-Cas 系统生成免疫记忆
- 批准号:
10231123 - 财政年份:2017
- 资助金额:
$ 43.55万 - 项目类别:
Generation of immunological memory by CRISPR-Cas systems
通过 CRISPR-Cas 系统生成免疫记忆
- 批准号:
9340801 - 财政年份:2017
- 资助金额:
$ 43.55万 - 项目类别:
Using CRISPR immunity to prevent the spread of virulence traits among pathogens
利用 CRISPR 免疫来防止病原体毒力特征的传播
- 批准号:
8356936 - 财政年份:2012
- 资助金额:
$ 43.55万 - 项目类别:
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