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.

项目总结 男性不育是一种复杂的病理情况，其特征是不同种类的 表型表现，使其根本原因不明。近年来，遗传性疾病不断涌现 作为男性不育的主要原因之一，至少占病例的15%。因此，理解 影响男性生育能力的各个方面的遗传网络，如精子发生(即精子 生产)对男性不育的诊断和治疗大有裨益。然而，估计 数千个基因可能参与精子发生，因此很难将特定的遗传原因归因于 男性不育。传统上，睾丸表达基因的功能可以通过产生基因敲除来分析。 小鼠品系被赋予了小鼠和人类精子发生的相似性。然而，这种方法 需要大量的时间和资源，这使得扩展具有挑战性。新兴技术，如 CRISPR筛选结合单细胞RNA测序(scRNA-seq)可以在规模上检查基因功能， 但在分析睾丸基因功能方面存在两个主要限制：(I)缺乏细胞培养模型 这忠实地概括了精子发生，这使得很难评估基因的扰动是否会导致 在体外产生精子的缺陷；以及(Ii)尽管可以读出基因扰动对细胞的内在影响 使用scrna-seq，由于组织的原因，基因扰动的细胞外效应不能被评估。 分离。这排除了使用CRISPR筛查来识别控制表型的基因 评估空间分辨率，如编码分泌因子的基因。因此，CRISPR屏幕 需要保留精子发生的空间背景的方法来询问睾丸基因功能 以很高的吞吐量。目前，开发空间分辨率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转录本；以及(Ii)读出 同时鉴定细胞内每个基因的扰动和转录产物。要解决这两个问题 主要挑战，我们将极大地改进和扩展我们最近的原位RNA测序协议 建立了直接在睾丸样本中对数百种信使核糖核酸进行空间分析的方法。我们还将表演一场 概念验证实验证实CRISPR引导RNA和mRNA在完整睾丸中共捕获 组织使用相同的原位测序方法。这些努力加在一起，将使高度创新的 功能基因组学方法在天然组织背景下以前所未有的方式剖析基因功能 空间分辨率和吞吐量。