Reducing Diversity at the Gamma Protocadherin Locus by CRISPR Targeting

描述(申请人提供)：哺乳动物神经系统的定义特征包括其神经细胞类型的巨大多样性以及它们在发育过程中相互作用形成数万亿突触的精致特异性。人们普遍认为，多个受体和细胞黏附分子(CAM)之间的组合作用提供的分子多样性是细胞识别的特异性所必需的。我们先前的工作已经确定由Pcdhg基因簇编码的22个伽马原钙粘附素(Gamma-Pcdhs)是提供这种分子多样性的首选候选者，因为它们介导的同亲反式相互作用是组合多样的(104-105)顺式四聚体。虽然对现有Pcdhg缺失小鼠的研究表明，这些分子对神经发育至关重要，但伽马-Pcdh异构体多样性在其功能中发挥作用的程度仍然是一个关键的未知问题，由于标准基因敲除小鼠生成的限制，该问题尚未得到解决。我们研究的长期目标是阐明分子多样性在调节神经发育关键方面的机制，这些关键方面与自闭症和智力残疾等人类疾病的病因有关。本申请的目的是利用CRISPR/CAS9技术 为了验证伽马-Pcdhs在体内的许多功能所需的异构体多样性的假说。我们将通过两个具体的目标来实现这一目标：1)使用CRISPR/Cas9系统来减少小鼠基因组中功能性Pcdhg亚型的分子多样性；2)确定在多大程度上需要伽马-Pcdhs的异构体多样性来满足其在神经发育中的已知作用。在对果蝇Dscam1基因的研究中，一个关键的突破是产生了交替外显子多样性降低的突变株。Dscam1基因可以通过组合选择性剪接产生19,008个不同的粘附异构体。对于小鼠的Pcdhg基因座，需要一种类似的方法，但需要新的、更高吞吐量的技术来产生突变系。最近描述的CRISPR-Cas9系统为同时靶向基因组中的多个位点产生插入突变提供了巨大的希望，这些突变将破坏基因。我们将针对22个Pcdhg可变外显子中的每一个设计CRISPR引导RNA序列，并将它们注射到受精的小鼠卵母细胞中，以生成一个小鼠品系的“库”，其中不同数量的这些外显子被破坏。这些株系中的一部分将被战略性地选择(涵盖广泛的完整Pcdhg外显子多样性)，并将对神经元存活、树突和轴突树枝形成以及突触发生的体内已知表型进行定量分析。通过这种方式，我们可以定义每种神经元表型依赖于伽马-Pcdh异构体多样性的程度，并更好地理解这些关键的CaM的作用机制。这项研究将进一步为CRISPR/Cas9系统同时进行基因打靶的特异性和有效性提供一个重要的体内测试，对促进小鼠反向遗传学的发展具有重要意义。

DESCRIPTION (provided by applicant): Defining characteristics of the mammalian nervous system include the vast diversity of its neuronal cell types and the exquisite specificity with which they interact to form trillions of synapses during development. It is widely believed that molecular diversity, provided by combinatorial interactions between multiple receptors and cell adhesion molecules (CAMs), is required for the specificity of cell recognition. Our prior work has identified the 22 gamma protocadherins (gamma -Pcdhs), encoded by the Pcdhg gene cluster, as top candidates for providing such molecular diversity because they mediate homophilic trans-interactions as combinatorially diverse (104-105) cis-tetramers. While studies on existing Pcdhg null mice demonstrate that these molecules are critical for neurodevelopment, the extent to which gamma -Pcdh isoform diversity plays a role in their functions remains a critical unknown that has not been addressed due to constraints of standard knockout mouse generation. The long- term goal of our research is to elucidate mechanisms of molecular diversity in regulating critical aspects of neurodevelopment relevant to the etiology of human disorders such as autism and intellectual disability. The objective of this application is to utilize CRISPR/Cas9 technology to test the hypothesis that isoform diversity of the gamma -Pcdhs is required for their many functions in vivo. We will pursue this objective through two Specific Aims: 1) Use the CRISPR/Cas9 system to reduce the molecular diversity of functional Pcdhg isoforms in the mouse genome; 2) Determine the extent to which the isoform diversity of gamma -Pcdhs is required for their known roles in neurodevelopment. In studies of the Drosophila Dscam1 gene, which can generate 19,008 distinct adhesive isoforms through combinatorial alternative splicing, a key breakthrough was the generation of mutant strains with reduced diversity of alternate exons. An analogous approach is needed for the Pcdhg locus in mice, but requires new, higher-throughput techniques for the generation of mutant lines. The recently- described CRISPR-Cas9 system holds great promise for the simultaneous targeting of multiple sites in the genome for the generation of indel mutations that will disrupt genes. We will design CRISPR guide RNA sequences targeting each of the 22 Pcdhg variable exons, and will inject them into fertilized mouse oocytes to generate a "library" of mouse lines in which varying numbers of these exons are disrupted. A subset of these lines will be strategically chosen (covering a wide range of intact Pcdhg exon diversity) and known in vivo phenotypes in neuronal survival, dendrite and axon arborization, and synaptogenesis will be analyzed quantitatively. In this way, we can define the extent to which each neuronal phenotype depends on gamma -Pcdh isoform diversity, and better understand the mechanisms through which these critical CAMs act. The proposed studies will further provide an important in vivo test of the specificity and efficiency o the CRISPR/Cas9 system for simultaneous gene targeting, of importance for the advancement of reverse genetics in the mouse.