Spatially Resolved CRISPR Genomics for Dissecting Testicular Gene Functions at Scale

空间分辨 CRISPR 基因组学用于大规模剖析睾丸基因功能

• 批准号: 10573701
• 负责人: Haiqi Chen
• 金额: $ 24.6万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10573701
• 关键词: Accounting; Address; Adopted; Affect; Bar Codes; CRISPR screen; CRISPR/Cas technology; Cell Culture Techniques; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Coupled; Couples; Coupling; Cultured Cells; Data; Data Set; Defect; Diagnosis; Emerging Technologies; Equipment and supply inventories; Exclusion; Fertility; Foundations; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genomic approach; Genomics; Guide RNA; Human; Image; Image Analysis; In Situ; In Vitro; Infertility; Knockout Mice; Label; Lentivirus; Male Infertility; Mediating; Messenger RNA; Methods; Modeling; Mus; Organ; Pathologic; Phenotype; Production; Protocols documentation; RNA Sequences; Reporter; Reproductive Biology; Research Personnel; Resolution; Resources; Sampling; Seminiferous tubule structure; Solid; Somatic Cell; Spermatogenesis; Testicular Tissue; Testis; Time; Tissues; Transcript; behavior influence; cell type; computational pipelines; design; experimental study; extracellular; functional genomics; gene function; genomic data; genomic tools; improved; in situ sequencing; in vivo; innovation; interest; knock-down; male; male fertility; next generation; sertoli cell; single-cell RNA sequencing; sperm cell; spermatogenic epithelium structure; stem cells; transcriptome; transcriptome sequencing; transcriptomic profiling

PROJECT SUMMARY Male infertility is a complicated pathological condition characterized by a heterogeneous spectrum of phenotypic presentations, rendering its underlying causes obscure. In recent years, genetic disorders emerge as one of the leading causes of male infertility, accounting for at least 15% of cases. Therefore, understanding the genetic network that influences various aspects of male fertility such as spermatogenesis (i.e., sperm production) would greatly benefit the diagnosis and treatment of male infertility. However, the estimate that thousands of genes may be involved in spermatogenesis makes it difficult to ascribe specific genetic causes to male infertility. Traditionally the functions of testis-expressing genes can be analyzed by generating knockout mouse lines given the similarities between mouse and human spermatogenesis. However, this approach demands significant time and resources, making it challenging to scale. Emerging technologies such as CRISPR screens coupled with single cell RNA sequencing (scRNA-seq) can examine gene functions at scale, but suffer from two major limitations for dissecting testicular gene functions: (i) the lack of a cell culture model that faithfully recapitulates spermatogenesis makes it difficult to assess whether perturbation of a gene leads to defects in sperm production in vitro; and (ii) while cell intrinsic effects of a gene perturbation may be read out using scRNA-seq, the extracellular effects of a gene perturbation cannot be assessed due to tissue disassociation. This excludes using CRISPR screens to identify genes controlling phenotypes that require spatial resolution to assess such as genes encoding for secreted factors. Therefore, a CRISPR screen approach that retains the spatial context of spermatogenesis is needed to interrogate testicular gene functions at a high throughput. There are currently two main challenges to develop a spatially resolved CRISPR screen approach: (i) to capture mRNA transcripts in situ at scale and at single-cell resolution; and (ii) to read out the identity of each gene perturbation and the mRNA transcripts within a cell simultaneously. To address these two main challenges, we will greatly improve and expand an in situ RNA sequencing protocol we have recently established to spatially profile hundreds of mRNA species directly in testicular samples. We will also perform a proof-of-concept experiment to demonstrate co-capture of CRISPR guide RNA and mRNA in intact testicular tissues using the same in situ sequencing approach. Together, these efforts will enable a highly innovative functional genomics approach to dissect gene functions in the native tissue context at an unprecedented spatial resolution and throughput.

项目摘要 男性不育是一种复杂的病理状态，其特征在于异质谱的 表型介绍，使其潜在的原因模糊。近年来，遗传性疾病的出现 作为男性不育症的主要原因之一，至少占病例的15%。因此了解 影响男性生育力的各个方面如精子发生的遗传网络（即，精子 生产）将大大有利于男性不育症的诊断和治疗。然而，据估计， 精子发生可能涉及数千个基因，因此很难将特定的遗传原因归因于 男性不育症传统的睾丸表达基因的功能分析方法是产生敲除基因 小鼠品系，因为小鼠和人类精子发生之间有相似之处。但是这种方法 需要大量的时间和资源，这使得扩展具有挑战性。等新兴技术 CRISPR筛选结合单细胞RNA测序（scRNA-seq）可以大规模检查基因功能， 但是在研究睾丸基因功能方面存在两个主要的限制：（i）缺乏细胞培养模型 忠实地再现精子发生使得难以评估基因的扰动是否会导致 体外精子产生缺陷;以及（ii）虽然基因干扰的细胞内在影响可能会被读出 使用scRNA-seq，基因扰动的细胞外效应不能被评估， 分离这排除了使用CRISPR筛选来鉴定控制表型的基因， 空间分辨率以评估例如编码分泌因子的基因。因此， 需要一种保留精子发生空间背景的方法来研究睾丸基因功能 以高吞吐量。目前开发空间分辨CRISPR屏幕面临两个主要挑战 方法：（i）以单细胞分辨率和规模原位捕获mRNA转录物;以及（ii）读出 在一个细胞内，每个基因扰动和mRNA转录物的同一性。为了解决这两 主要挑战，我们将大大改善和扩大原位RNA测序协议，我们最近 建立了直接在睾丸样品中对数百种mRNA进行空间分析的方法。我们还将执行一个 证明CRISPR指导RNA和mRNA在完整睾丸中的共捕获的概念验证实验 使用相同的原位测序方法。总之，这些努力将使高度创新的 功能基因组学方法，以前所未有的速度在天然组织背景下剖析基因功能， 空间分辨率和吞吐量。