CRISPR interference of essential stage-specific gene function in Chlamydia trachomatis

CRISPR干扰衣原体关键阶段特异性基因功能 沙眼衣原体 摘要 沙眼衣原体是临床上重要的专性细胞内细菌病原体， 可预防的传染性失明（沙眼）和细菌性性传播疾病的主要原因 世界各地的疾病。如果不清除，那么衣原体感染会导致健康状况不佳 后果，如盆腔炎和输卵管因素的妇女不孕。尽管 它对人类健康的破坏性影响，大部分C。沙眼的病理生物学仍然未知。 而C.沙眼研究得很好， 监管途径和机制仍然不明确，破坏了基本的 理解C。沙眼发病机制学习基本知识是非常具有挑战性的， 基因，其破坏导致致命性。为了弥补这一差距，我们成功地调整了 CRISPR干扰（CRISPRi）基因沉默技术，它依赖于诱导型表达 抑制基因转录的dCas直向同源物的催化失活变体， 染色体位点。沙眼这种方法允许创建有条件的、可逆的 敲除单个基因，也可以通过包括质粒编码的 作为与dCas直向同源物的转录融合的靶基因的拷贝。根据这些 研究发现，我们进一步调整了我们的CRISPRi方法， 敲低用于同时靶向敲低多个基因。的目的 一项新的研究计划是开发下一代CRISPR干扰（CRISPRi）基因编辑。 工具箱C沙眼，并探讨其对阐明潜在机制的效用 发育基因调控的关键。在具体目标1中，我们将建立一个适应性强的第二个目标， CRISPRi平台的生成，可实现可调，可跟踪和定量评估 CRISPRi介导的C.沙眼衣原体的细胞和分子表型。 在具体目标2中，我们将测试我们的假设，即一组“已知”和“未知”基因是 对网状体（RB）的终止复制和RB分化的关键事件至关重要 传染性基本体（EB）。利用CRISPRi，这个目标将阐明相对的 每个必需基因产物对C.沙眼发育过程至关重要 用于EB形成和重新开始新的感染周期。总的来说，这项创新的研究 该项目将寻求一个重点调查，以说明衣原体因素是至关重要的， 这些因素是如何直接影响C. 沙眼这一点也很重要，因为所制定的方法将是 对C.沙眼

CRISPR interference of essential stage-specific gene function in Chlamydia trachomatis Abstract Chlamydia trachomatis is a clinically important obligate intracellular bacterial pathogen and the leading cause of preventable infectious blindness (trachoma) and bacterial sexually transmitted diseases worldwide. If not cleared, then chlamydial infections can lead to poor health consequences, such as pelvic inflammatory disease and tubal factor infertility in women. Despite its devastating impacts on human health, much of C. trachomatis pathobiology remains unknown. Whereas the characteristics of the developmental cycle of C. trachomatis are well studied, the regulatory pathways and mechanisms remain poorly defined, undermining fundamental understanding of C. trachomatis pathogenesis. It is extremely challenging to study essential genes, whose disruption leads to lethality. To address this gap, we have successfully adapted CRISPR interference (CRISPRi) gene silencing technology, which relies on inducible expression of catalytically inactive variants of dCas orthologs that repress gene transcription at a specific chromosomal locus in C. trachomatis. This approach allows for creation of conditional, reversible knockdowns in individual genes that can also be complemented by including a plasmid-encoded copy of the targeted gene as a transcriptional fusion with the dCas ortholog. Building on these findings, we have further adapted our CRISPRi approaches to allow for multiplexed gene knockdown for the targeted knockdown of multiple genes simultaneously. The objective of the proposed study is to develop a next generation of CRISPR interference (CRISPRi) genetic editing toolbox in C. trachomatis and to explore its utility towards elucidating the underlying mechanisms of developmental gene regulation. In Specific Aim 1, we will establish an adaptable second generation of CRISPRi platform, which enables tunable, trackable, and quantitative assessment of CRISPRi-mediated changes in C. trachomatis cellular and molecular phenotypes in single cells. In Specific Aim 2 we will test our hypothesis that a set of “known” and “unknown” genes are essential for critical events of ending replication of reticulate bodies (RB) and RB differentiating to the infectious elementary body (EB). Leveraging CRISPRi, this Aim will elucidate the relative contributions of each essential gene product to C. trachomatis developmental processes critical for EB formation and reinitiation of a new cycle of infection. Collectively, this innovative research project will seek a focused investigation to illustrate what chlamydial factors are crucial for, and how these factors directly contribute to, regulation of the late developmental cycle of C. trachomatis. It is also significant, as the methods developed will be the starting point towards interrogation of genome-wide studies of gene function and genetic interactions in C. trachomatis.