RNA-programmable cell-type targeting, editing, and therapy

Josh Huang 2020年9月6日 RNA可编程细胞类型靶向、编辑和治疗 摘要 细胞类型的系统识别和操作对于解剖生物学的机制是必要的。 在健康和疾病中发挥作用。虽然大规模的单细胞转录组分析现在可以 鉴定许多生物体中所有主要的转录定义的细胞类型， 需要获得所有主要的细胞群，以在生理学和解剖学上验证这些统计学结果。 “转录簇”作为细胞类型，更重要的是，询问他们在组织组织中的作用， 功能选择性操纵细胞类型的困难仍然是此类研究的关键障碍。电流 解决这个问题的方法主要依赖于生殖系DNA工程，这是缓慢和昂贵的， 伦理问题，特别是在人类和其他灵长类动物中。细胞型转录增强子提供了一个非- 目前，已经有许多研究采用了种系方法，但它们的鉴定和验证仍然需要大量的努力和昂贵的费用。克服这些 障碍，所有的生物医学研究迫切需要一种新的方法来操纵细胞类型的方式， 具体，简单而全面，负担得起，并在器官和物种之间推广，类似于CRISPR- 基于基因的操纵。在这里，我建议开发一种范式转换技术，使RNA- 基于RNA编辑的基础生物学的可编程细胞类型靶向和操纵。实现 这一突破，我将利用一套下一代，多功能核糖核蛋白设备，它可以 检测体细胞中特定RNA的存在并触发细胞效应基因的表达， 可视化、监控和操作。这种方法建立在通用RNA传感和编辑的基础上。 所有后生动物细胞内的系统，以作用于RNA的编辑酶腺苷脱氨酶为中心 （阿达尔）。我将这种方法称为CellREADR（通过内源性阿达尔的RNA传感的细胞进入）。作为 CellREADR利用内源性细胞机制，由单个模块化RNA分子构建， 功能通过沃森克里克基地配对，它是高度具体的，固有的可编程，快速，负担得起的，容易 使用，并广泛适用。我建议在细胞培养系统中构建和优化CellREADR设备， 在一个高度复杂的器官-大脑-通过靶向和操纵大量的神经元， 小鼠大脑皮层中的细胞类型。我们将通过靶向细胞类型， 体内人脑标本，以及猕猴和鸟类大脑。此外，我们将设计交叉 靶向越来越多的特定细胞类型的策略，以及同时和 组织中多种细胞类型的差异化操作。通过将细胞型RNA传感器连接到各种效应器， 改变细胞功能的基因，从消融到微妙的生理调节，我们的目标是编辑细胞 下一代组织工程的组成和功能。这项技术将导致大量的 CellREADR文库用于靶向不同物种的所有主要细胞类型，类似于基于CRISPR的基因编辑 多样的基因组。因此，CellREADR将在基础生物学、医学和生物技术领域产生广泛的影响。

Josh Huang Sept 6, 2020 RNA-programmable cell-type targeting, editing, and therapy Abstract Systematic identification and manipulation of cell types is necessary for dissecting mechanisms of biological functions in health and disease. Although large-scale, single-cell transcriptome profiling now enables identification of all major transcription-defined cell types in many organisms, easy and systematic experimental access to all major cell populations is needed to physiologically and anatomically validate these statistical “transcriptional clusters” as cell types and, more importantly, to interrogate their roles in tissue organization and function. The difficulty of selectively manipulating cell types remains a critical barrier to such studies. Current approaches to this problem mostly rely on germline DNA engineering, which is slow and expensive and poses ethical issues, especially in humans and other primates. Cell-type transcriptional enhancers afford a non- germline approach, but their identification and validation remain effort-intensive and costly. To overcome these barriers, all of biomedical research urgently needs a novel approach to manipulate cell types in a way that is specific, easy yet comprehensive, affordable, and generalizes across organs and species, akin to CRISPR- based manipulation of genes. Here I propose to develop a paradigm-shifting technology that will enable RNA- programmable cell-type targeting and manipulation based on the fundamental biology of RNA editing. To achieve this breakthrough, I will harness a set of next-generation, multi-functional ribonucleoprotein devices, which can detect the presence of specific RNAs in somatic cells and trigger the expression of effector genes for cell visualization, monitoring, and manipulation. This method builds upon the universal RNA sensing and editing system within all metazoan cells, centered around the editing enzyme adenosine deaminase acting on RNA (ADAR). I term this method CellREADR (Cell access through RNA sensing by Endogenous ADAR). As CellREADR leverages endogenous cellular machinery and is built with a single modular RNA molecule that functions through Watson-Crick base pairing, it is highly specific, inherently programmable, fast, affordable, easy to use, and widely applicable. I propose to build and optimize CellREADR devices in cell-culture systems and validate the method in a highly complex organ - the brain - by targeting and manipulating a large set of neuronal cell types in the mouse cerebral cortex. We will extend CellREADR across species by targeting cell types in ex vivo human brain specimens, and in the macaque and avian brain. Further, we will design intersectional strategies for targeting increasingly specific cell types, and combinatorial strategies for simultaneous and differential manipulation of multiple cell types in a tissue. By linking cell-type RNA sensors to a variety of effector genes that alter cell functions, ranging from ablation to subtle physiological modulation, we aim to edit cell composition and function for next-generation tissue engineering. This technology will result in large arrays of CellREADR libraries for targeting all major cell types across diverse species, akin to CRISPR-based gene editing of diverse genomes. Thus, CellREADR will have a broad impact in basic biology, medicine, and biotechnology.