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 男性不育症是一种复杂的病理状况，其特征是 表型演示，使其潜在的呈现会导致晦涩。近年来，遗传疾病出现 作为男性不育症的主要原因之一，占案件的至少15％。因此，理解 影响男性生育能力的各个方面的遗传网络，例如精子发生（即精子 生产）将极大地有利于男性不育症的诊断和治疗。但是，估计 数以千计的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 男性不育症。传统上，可以通过产生敲除可以分析表达睾丸基因的功能 小鼠线给出了小鼠和人类精子发生之间的相似性。但是，这种方法 需要大量的时间和资源，使其构成挑战。新兴技术，例如 CRISPR筛选以及单细胞RNA测序（SCRNA-SEQ）可以大规模检查基因功能， 但是，剖析睾丸基因功能有两个主要局限性：（i）缺乏细胞培养模型 忠实地概括了精子发生，因此很难评估基因扰动是否导致 体外精子产生缺陷； （ii）虽然可以读出基因扰动的细胞固有效应 使用SCRNA-SEQ，由于组织无法评估基因扰动的细胞外影响 分离。这不包括使用CRISPR屏幕来识别控制需要表型的基因 空间分辨率评估诸如编码分泌因子的基因之类的。因此，CRISPR屏幕 需要保留精子发生空间上下文的方法来询问睾丸基因功能 在高吞吐量。目前有两个主要的挑战来开发空间解决的CRISPR屏幕 方法：（i）以大规模和单细胞分辨率捕获mRNA转录本； （ii）读出 每个基因扰动的身份和细胞内的mRNA转录物的身份同时。解决这两个 主要挑战，我们将大大改善和扩展我们最近拥有的原位RNA测序协议 在睾丸样品中直接在空间上介绍了数百种mRNA物种。我们还将执行 概念验证实验，以证明完整睾丸中CRISPR指南RNA和mRNA的共同捕获 使用相同的原位测序方法的组织。这些努力共同使高度创新的 功能基因组学方法是在天然组织环境中剖析基因功能在前所未有的 空间分辨率和吞吐量。