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.

Public Health Statement The mammalian transcriptome regulates numerous critical cell functions, and the ability to con- trol the transcriptome will lead to breakthrough cellular therapies for many diseases. We pro- posed to develop tools that can enhance the sensitivity and specificity of transcriptome engi- neering and demonstrate their clinical potential by improving the safety of cell therapy. Success from this work will greatly enhance our ability to design novel therapeutic and diagnostic appli- cations.