Advancing programmable RNA-targeting tools for research and therapeutics

项目摘要 我们之前开发了一套基于RNA靶向CRISPR的调节和研究RNA的工具， Cas13系统已被许多生命科学领域的研究人员采用或扩展。这项建议 旨在发现和表征额外的可编程RNA结合蛋白，并开发它们 用于分子技术。特别是，我们专注于识别超小Cas13蛋白， 它可以与RNA编辑效应子融合，为精确的单碱基转录物创造紧凑的平台， 编辑. RNA编辑在一系列疾病中具有显著的治疗潜力，包括遗传性疾病。 编辑基因组不可能或风险太大的疾病，以及短暂遗传损伤 改变是可取的。因此，另一个目的是证明使用RNA编辑治疗的可行性。 除了RNA编辑器，我们还将使用我们识别的新蛋白质来开发转录状态传感器， 可用于标记异源群体中的特定细胞亚型，用于成像、分离或 功能性成果。 为了实现这些目标，我们将利用我们以前的经验来发现和表征新的RNA 针对CRISPR系统，重点是识别支持RNA编辑活性的小酶。我们将 还探索了在我们的RNA编辑构建体中使用新型RNA脱氨酶的可能性。除了 创建RNA编辑构建体，我们还将RNA靶向酶融合到GFP或Cre中， 转录传感器我们工作的一个关键方面将是蛋白质工程。候选酶（或其 RNA组分）可能需要被修饰以在哺乳动物细胞中具有有效的特异性活性。我们将使用蛋白质 工程化以增加RNA脱氨酶的特异性和活性，以及扩展底物基础 这些酶的偏好。对于我们的转录状态传感器，蛋白质工程将是核心， 成功地产生提供高特异性和高信噪比的传感器。所有这些努力将 以相关酶的结构和生物化学研究为指导。 最后，我们将在急性肝损伤的小鼠模型中应用紧凑的高特异性RNA编辑器， 证明其作为短期可逆治疗的治疗潜力。同时，我们将演示 使用RNA编辑来纠正导致神经发育障碍Rett综合征的突变的可行性 用的是以前建立的这种疾病的小鼠模型。 这项工作将大大推进RNA编辑的临床应用，并揭示有关RNA编辑的新生物学。 CRISPR系统和其他微生物防御系统。

PROJECT SUMMARY We previously developed a suite of tools for modulating and studying RNA based on the RNA-targeting CRISPR- Cas13 system, which has been adopted or extended by many researchers in the life sciences. This proposal seeks to discover and characterize additional programmable RNA binding proteins and develop them for use as molecular technologies. In particular, we are focusing on identifying ultra-small Cas13 proteins, which can be fused to RNA editing effectors to create compact platforms for precision, single-base transcript editing. RNA editing has significant therapeutic potential across a spectrum of conditions, including genetic diseases where it is not possible or too risky to edit the genome as well as acute insults where transient genetic changes are desirable. Thus, another aim is to demonstrate the feasibility of using RNA editing therapeutically. Beyond RNA editors, we will also use the new proteins we identify to develop transcriptional state sensors, which can be used to mark specific cell sub-types within a heterogenous population, either for imaging, isolation, or functional outcomes. To achieve these goals, we will leverage our previous experience to discover and characterize new RNA targeting CRISPR systems, with a focus on identifying small enzymes that support RNA editing activity. We will also explore the possibility of using novel RNA deaminase enzymes in our RNA editing constructs. In addition to creating RNA editing constructs, we will also fuse the RNA targeting enzymes to GFP or Cre to create transcriptional sensors. A critical aspect of our work will be protein engineering. Candidate enzymes (or their RNA components) may need to be modified for efficient, specific activity in mammalian cells. We will use protein engineering to increase the specificity and activity of RNA deaminases, as well as extend the substrate base preference of these enzymes. For our transcriptional state sensors, protein engineering will be central to successfully generating sensors that afford high specificity and high signal-to-noise ratios. All of these efforts will be guided by structural and biochemical studies of the relevant enzymes. Finally, we will apply the compact, high-specificity RNA editors in a mouse model of acute liver damage to demonstrate their therapeutic potential as short-lived, reversible treatments. In parallel, we will demonstrate the feasibility of using RNA editing to correct a mutation that causes the neurodevelopmental disorder Rett syndrome using a previously established mouse model of this disease. This work will substantially advance RNA editing toward clinical use, as well as uncover new biology about CRISPR systems and other microbial defense systems.