CRISPR/Cas9 and small molecules for targeting sperm function and fertilization

项目2摘要(CRISPR/Cas9和用于靶向精子功能和受精的小分子) 项目2的总体目标是使用CRISPR/Cas9来了解功能性精子的形成 并确定以受精所需蛋白质为靶点的小分子探针和临床前候选蛋白质 以达到体内的避孕效果。人口增长是一个重大的世界性问题，我们的资源不能 继续保持这些人口增长，正因为如此，NICHD已将避孕作为优先事项 焦点区域。因为我们小组致力于开发专门针对精子的避孕药 而受精，这些生殖细胞选择性避孕药将消除不想要的副作用。伊卡瓦人 实验室已经开发出CRISPR/Cas9系统，可以在体内有效地突变基因，包括Cetn1和 男性生育所需的PRM1基因。在本次P01中，我们将使用CRISPR/Cas9技术来推进我们的 了解所有三个项目中的生育途径，并产生有用的信息来推动这些目标 蛋白质和其他相关途径蛋白进入DEC-Tec核心进行筛选。年的避孕目标 项目2，我们将重点研究两个精子-卵子融合跨膜/信号蛋白和四个精子运动- 我们已经展示的相关蛋白质在进化上是保守的，只对男性生育能力是必需的。伊卡瓦人 实验室已经有效地利用CRISPR/Cas9系统在200多个小鼠基因中产生了突变 包括这项提案中描述的基因。这一战略也成功地产生了TAG的敲门砖 将序列转换成基因座。我们独立使用CRISPR/Cas9，并与Matzuk合作使用 实验室(项目1)发现精子-卵子融合和精子运动所需的蛋白质。功能分析 这些小鼠模型不仅有助于破译这些蛋白质在体内发挥作用的机制 精子-卵子融合和精子运动，也是开发针对这些关键的避孕药 小路。使用DEC-TEC Core筛选小分子将加速这一过程。我们的整体 假设CRISPR/CAS9和DEC-Tec将帮助我们理解这些相互作用的网络 精子功能的蛋白质，快速识别针对这些必需的多个小分子 生精蛋白，并为男性和女性创造了一种避孕药具。的具体目标 项目2是：1)操纵小鼠基因组并描述精子-卵子融合的相互关系 蛋白质；2)使用CRISPR/Cas9研究新的睾丸特异蛋白质的作用机制；3)使用 DEC-Tec确定小分子类药物探针和临床前候选药物以抑制精子运动特异性 以及体内具有避孕效果的受精特异蛋白质。项目2的成功和这笔P01赠款 依靠项目2与项目1中的Matzuk博士、Sonnenburg博士、Lamb博士和 项目3中的黄，以及DEC-Tec核心中的科学家。我们的目标是开发出多种独特的转基因 在这项P01资助中，用于体内机械学和药理学研究的模型和非激素避孕药。

PROJECT 2 SUMMARY (CRISPR/Cas9 and small molecules for targeting sperm function and fertilization) The overall goals of Project 2 are to use CRISPR/Cas9 to understand the formation of functional sperm and identify small-molecule probes and preclinical candidates to target fertilization-required proteins for a contraceptive effect in vivo. Population growth is a major worldwide issue, and our resources cannot continue to sustain these population increases, and because of this, NICHD has made contraception a priority focus area. Because our group is focused on the development of contraceptives that specifically target sperm and fertilization, these germ cell-selective contraceptives would eliminate unwanted side effects. The Ikawa laboratory has developed the CRISPR/Cas9 system to efficiently mutate genes in vivo including the Cetn1 and Prm1 genes required for male fertility. In this P01, we will use CRISPR/Cas9 technology to advance our understanding of fertility pathways in all three Projects and generate useful information to move these target proteins and other related pathway proteins into the DEC-Tec Core for screening. For contraceptive targets in Project 2, we will focus on two sperm-egg fusion transmembrane/signaling proteins and four sperm motility- related proteins that we have shown are evolutionarily-conserved and required only for male fertility. The Ikawa laboratory has effectively used the CRISPR/Cas9 system to generate mutations in over 200 mouse genes including the genes described in this proposal. This strategy has also successfully produced knockin of tag sequences into loci. We have used CRISPR/Cas9 independently and in collaboration with the Matzuk laboratory (Project 1) to uncover proteins required for sperm-egg fusion and sperm motility. Functional analysis of these mouse models will help not only to decipher the mechanisms by which these proteins function in sperm-egg fusion and sperm motility but also to develop contraceptive drugs to target these essential pathways. The screening of small molecules using the DEC-TEC Core will accelerate this process. Our overall hypothesis is that CRISPR/Cas9 and DEC-Tec will help us to understand the network of interactions of these proteins for sperm function, rapidly identify multiple small molecules that are directed at these essential spermatogenic proteins, and create an assortment of contraceptives for men and women. The Specific Aims of Project 2 are: 1) Manipulate the mouse genome and characterize the interrelationship of sperm-egg fusion proteins; 2) Use CRISPR/Cas9 to study the mechanism of action of novel testis-specific proteins; and 3) Use DEC-Tec to identify small-molecule drug-like probes and preclinical candidates to inhibit sperm motility-specific and fertilization-specific proteins for a contraceptive effect in vivo. The success of Project 2 and this P01 grant relies on the continued collaborations of Project 2 with Dr. Matzuk in Project 1, Drs. Sonnenburg, Lamb, and Huang in Project 3, and the scientists in the DEC-Tec Core. Our goal is to develop multiple unique transgenic models and non-hormonal contraceptives for mechanistic and pharmacologic studies in vivo in this P01 grant.