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.

项目概要 男性不育症是一种复杂的病理状况，其特征为异质谱 表型呈现，使其根本原因变得模糊。近年来，遗传性疾病不断出现 作为男性不育的主要原因之一，至少占病例的 15%。因此，了解 影响男性生育力各个方面的遗传网络，例如精子发生（即精子） 生产）将极大地有益于男性不育症的诊断和治疗。然而，估计 数千个基因可能与精子发生有关，因此很难将特定的遗传原因归因于 男性不育症。传统上可以通过敲除来分析睾丸表达基因的功能 小鼠品系给出了小鼠和人类精子发生之间的相似性。然而，这种方法 需要大量的时间和资源，使得扩展具有挑战性。新兴技术，例如 CRISPR 筛选与单细胞 RNA 测序 (scRNA-seq) 相结合可以大规模检查基因功能， 但解剖睾丸基因功能面临两个主要限制：（i）缺乏细胞培养模型 忠实地再现精子发生使得很难评估基因的扰动是否会导致 体外精子生成缺陷； (ii) 可以读出基因扰动的细胞内在效应 使用 scRNA-seq，由于组织的不同，无法评估基因扰动的细胞外效应 分离。这排除了使用 CRISPR 筛选来识别控制表型的基因，这些基因需要 空间分辨率来评估例如编码分泌因子的基因。因此，CRISPR筛选 需要保留精子发生空间背景的方法来询问睾丸基因功能 在高吞吐量下。目前开发空间分辨 CRISPR 筛选面临两个主要挑战 方法：(i) 以单细胞分辨率原位捕获 mRNA 转录物； (ii) 读出 同时识别细胞内每个基因扰动和 mRNA 转录本。为了解决这两个问题 主要挑战，我们将极大地改进和扩展我们最近的原位 RNA 测序方案 建立直接在睾丸样本中对数百种 mRNA 进行空间分析。我们还将进行一次 概念验证实验证明 CRISPR 引导 RNA 和 mRNA 在完整睾丸中的共同捕获 使用相同的原位测序方法对组织进行测序。这些努力共同将实现高度创新 功能基因组学方法以前所未有的方式剖析天然组织背景中的基因功能 空间分辨率和吞吐量。