Cas9 RNP targeting cis-regulatory elements in lymphocytes

项目摘要 用于基因组编辑的CRISPR工具彻底改变并加速了编码的功能性询问 在模型动物系统和人体细胞中的基因，包括初级淋巴细胞， 在过敏和哮喘的免疫病理学中的作用。我们建议调整和优化这些用于miRNA的工具- 定向路径发现首先，将为我们现有的目标创建合并的逆转录病毒CRISPR文库， 根据经验鉴定了miR-29，一种在T细胞和B细胞中具有关键功能的miRNA。二是 将下一代基于转染的CRISPR系统扩展到靶向顺式调控序列， 编码蛋白质，但介导miRNA和靶mRNA之间的调节相互作用。成功在这些 目标将使详细的机制分析的miRNA：目标网络，管理免疫反应， 哮喘和过敏。该提案的第二个重要目标是支持职业和科学 塞莱斯特加尔萨的发展，一个学士后研究员获得研究经验，作为一个大三 安塞尔实验室的专家拟议中的实验建立在她在安塞尔实验室学到的技能之上， 并将为她提供机会，以发展研究叙述和科学成就， 申请明年的博士课程。

Project Summary CRISPR tools for genome editing have revolutionized and accelerated the functional interrogation of coding genes in model animal systems and in human somatic cells, including primary lymphocytes that play critical roles in the immunopathology of allergy and asthma. We propose to adapt and optimize these tools for miRNA- directed pathway discovery. First, will create pooled retroviral CRISPR libraries for targets that we have empirically identified for miR-29, a miRNA with critical functions in both T cells and B cells. Second, we will extend next generation transfection-based CRISPR systems to target cis-regulatory sequences that do not code for protein, but mediate regulatory interactions between miRNAs and target mRNAs. Success in these aims will enable detailed mechanistic analysis of miRNA:target networks that govern immune responses in asthma and allergy. A second important goal of this proposal is to support the career and scientific development of Celeste Garza, a post-baccalaureate researcher gaining research experience as a junior specialist in the Ansel laboratory. The proposed experiments build on skills she has learned in the Ansel lab, and will provide the opportunity for her to develop a research narrative and scientific achievements as prepares to apply for PhD programs in the coming year.