Multiplexed and Logical Control of the Mammalian Transcriptome Using Cas13

哺乳动物的转录组整合了不同的细胞外和细胞内信号，并控制着许多 关键的细胞功能。此外，灵活控制内源基因表达的能力 外部和内部信号将导致具有更高安全性和有效性的突破性细胞疗法。然而， 我们缺乏可逆地、有效地调节转录组的工具。基因组编辑工具，RNA内部 Ference和可编程转录因子是强大的基因工程工具。然而，他们都有 一些缺陷，要么是它们永久性地扰乱了感兴趣的基因，而后者阻止了对死亡原因的研究。 基因的动态影响，或者它们缺乏特异性和活性。因此，迫切需要新的工具来 提高我们对哺乳动物转录组重新编程的能力。 最近，一类新的RNA引导的RNA核酸酶--Cas13被发现。一些Cas13的同源基因， 如Cas13d和Cas13b，已被证明具有比 哺乳动物细胞中的RNAi。Cas13也不受染色质结构的影响，这一挑战通常包括- 通过可编程转录因子进行转录组工程。它也没有抵押品 在哺乳动物细胞中具有其他Cas13所具有的切割活性。因此，Cas13有潜力成为最好的 哺乳动物转录组工程的工具。 与其他基因组工程工具类似，Cas13的全部潜力只有在调控机制- NIMS和基因电路已经被结合在一起，提供了便捷和智能的控制。目前，有以下几种 没有专门设计的工具来规范Cas13的活动。因此，这项拟议工作的目标是 用Cas13开发一套突破性的工具，可以诱导和可逆地调节哺乳动物 对外源小分子(和FDA批准的药物)或生物提示做出反应的转录组。此外，还可以 展示他们的临床潜力，我们将利用我们设计的Cas13来提高嵌合抗原的安全性 受体(CAR)T细胞治疗。CAR T细胞疗法已显示出巨大的前景和对VARI的疗效。 但它们也可能导致细胞因子释放综合征(CRS)，这是一种危险的不良副作用。这个 CAR T细胞产生的导致CRS的因子也是该疗法发挥作用所必需的。因此， 需要对这些关键因素进行可诱导和可调整的控制，而不是击倒，以开发安全和 有效的CAR T细胞治疗。我们将通过以下目标实现我们的目标： 目标1：开发可分离诱导的Cas13集合 目的2：响应生物信号建立Cas13的细胞自主控制 目的3：调节CAR T细胞中细胞因子的产生以对抗CRS 这项工作的成功将对细胞疗法的发展产生变革性的影响。

The mammalian transcriptome integrates diverse extracellular and intracellular signals and controls numerous critical cell functions. Furthermore, the ability to flexibly control endogenous gene expression in response to external and internal signals will lead to breakthrough cell therapies with enhanced safety and efficacy. However, we lack tools that can reversibly and effectively modulate the transcriptome. Genome editing tools, RNA inter- ference, and programmable transcription factors are powerful genetic engineering tools. However, they all have some deficiencies, either they permanently disrupt the gene of interest, which prevents investigation of the dy- namic impact of the gene, or they lack specificity and activity. Therefore, novel tools are urgently needed to advance our ability to reprogram the mammalian transcriptome. Recently, a new class of RNA-guided RNA nuclease, Cas13, has been discovered. Some Cas13 orthologs, such as Cas13d and Cas13b, have been shown to have higher and more specific RNA degradation activity than RNAi in mammalian cells. Cas13 is also not affected by the chromatin structure, a challenge commonly encoun- tered by programmable transcription factors for transcriptome engineering. It also doesn't have the collateral cleavage activity in mammalian cells that other Cas13 has. Therefore, Cas13 has the potential to be the best tool for mammalian transcriptome engineering. Similar to other genome engineering tools, the full potential of Cas13 can only be realized if regulatory mecha- nisms and genetic circuits have been incorporated that afford facile and intelligent control. Currently, there are no tools specifically designed to regulate Cas13 activity. Therefore, the objective of this proposed work is to develop a set of groundbreaking tools with Cas13 that can inducibly and reversibly modulate the mammalian transcriptome in response to exogenous small molecules (and FDA approved drugs) or biological cues. Also, to showcase their clinical potential, we will leverage our engineered Cas13 to improve the safety of chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapies have shown tremendous promise and efficacy against vari- ous cancers, but they can also lead to cytokine release syndrome (CRS), a dangerous adverse side effect. The factors produced by CAR T cells lead to CRS are also necessary for the function of the therapy. Therefore, inducible and tunable control of these critical factors, as opposed to knockout, are required to develop safe and effective CAR T cell therapy. We will achieve our objectives through the following aims: Aim 1: Develop a collection of split inducible Cas13 Aim 2: Establish cell-autonomous control of Cas13 in response to biological signals Aim 3: Modulate cytokine production in CAR T cells to combat CRS Success from this work will have a transformative impact on cell therapy development.