FET: Small: Scalable transcriptional programs through feedback regulation

对活细胞进行可靠编程的能力将对许多应用产生巨大影响。例如，在生物传感和生物修复中，工程微生物将传感和降解环境污染物或病原体；在个性化医疗中，患者来源的细胞将被重新编程为感兴趣的细胞类型，以治疗各种疾病；在人类的表现中，可编程益生菌可以感知压力和疲劳水平，并释放缓解药物。编程生物学要实现这些复杂的功能，需要复杂的计算来选择性地分类环境线索，处理它们，并使适当的细胞反应。增加的复杂性通常需要增加实现功能的逻辑程序的规模。为了扩大一个人可以在活细胞中编码的遗传程序的规模，需要许多部分互不干扰。虽然可以设计许多正交部分，但它们通常需要一个公共资源来实现其指定的激活或抑制功能。最近才变得明显的是，由于这个公共资源的负载，增加的规模通常会损害功能，因为单个部件的资源数量将不足。这个项目的目标是解决这个问题，从而实现可扩展的转录程序。具体而言，本项目将创建dCas9水平的反馈控制器，dCas9水平是许多遗传部分共享的蛋白质资源，它补偿负载效应，并将所需的dCas9蛋白量分配到各个子系统。因此，可以在不影响功能的情况下增加系统规模。反过来，这将使达到具体应用所需的系统功能的复杂程度和可靠性成为可能。该项目最终将产生一个广泛适用的工具，许多实验室将能够很容易地使用dCas9实现真正可扩展的转录程序。该项目将支持控制设计和合成生物学交叉方向的研究生和本科生的跨学科教育。教育和推广机制将包括研究监督，教授生物分子反馈系统课程，包括该项目的材料，组织一个研讨会，将控制理论和合成生物学社区聚集在一起，并在剑桥科学节上展示一个关于遗传电路的展位。该项目的最终目标是通过确保组合逻辑门在程序中包含其他门时保持其预期功能，从而实现CRISPR/dCas9转录程序的稳健性，模块化和可扩展性。工程CRISPR/dCas9系统是创建大规模转录程序的有前途的工具，因为人们可以设计单引导rna （sgrna）将dCas9靶向到任何所需的DNA序列上，以执行转录抑制（CRISPRi）或激活（CRISPRa）。这允许，原则上，一个无限的正交部分库。然而，很明显，电路中sgRNA数量的增加降低了每个sgRNA抑制其靶标的能力，因为sgRNA负载dCas9，当dCas9与一个sgRNA结合时，其他sgRNA无法使用它。这种加载现象耦合了理论上独立的门，导致了严重的性能下降。为了解决这些问题，有必要设计工程方法来减轻dCas9负载的影响。本项目将设计可调节的dCas9发生器，其中一个反馈控制器将未绑定的dCas9电平调节到一个恒定值，而不依赖于gRNA负载的存在。抑制电路将首先考虑，激活电路以及混合电路将在第二步解决。稳压dCas9发生器的性能将在越来越复杂的分层逻辑门（包括级联和NAND门）上得到验证。这些调节发电机将确保当系统中包含额外的门时，任何一个门的功能都被保留。最终，使用受调控的dCas9发生器将增加CRISPR/dCas9程序的规模、复杂性和可靠性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The ability to reliably program living cells is going to have tremendous impact on a number of applications. For example, in biosensing and bioremediation, engineered microorganisms will sense and degrade environmental pollutants or pathogens; in personalized medicine, patient-derived cells will be reprogrammed to a cell type of interest to cure various diseases; in human performance, programmable probiotics will sense stress and fatigue level and release mitigating drugs. Programming biology to achieve these sophisticated functionalities requires complex computation to selectively classify environmental cues, process them, and enable appropriate cellular responses. Increased complexity often requires increased scale of the logic programs that implement function. To augment the scale of the genetic programs that one can encode in living cells, many parts are required such that they do not interfere with one another. Although, many orthogonal parts can be designed, they often require a common resource for implementing their specified activation or repression function. It has become apparent only recently that, because of loads to this common resource, increased scale will often compromise functionality since individual parts will have insufficient amount of resources. The goal of this project is to address this problem, thus enabling scalable transcriptional programs. Specifically, this project will create feedback controllers of dCas9 level, a protein resource shared among many genetic parts, which compensate for load effects and distribute the required amount of dCas9 protein to each subsystem. As a consequence, increased system scale will be possible without compromising functionality. This, in turn, will enable reaching the level of sophistication and reliability of system’s function needed for concrete applications. The project will ultimately produce a broadly applicable tool that many labs will be able to readily use to achieve truly scalable transcriptional programs using dCas9. This project will support interdisciplinary education of graduate and undergraduate students at the cross-roads of control design and synthetic biology. Education and outreach mechanisms will include research supervision, teaching a biomolecular feedback systems course including materials from this project, organizing a workshop bringing control theory and synthetic biology communities together, and presenting a booth on genetic circuits at the Cambridge Science Festival. The ultimate objective of this project is to enable robustness, modularity, and hence scalability of CRISPR/dCas9 transcriptional programs by ensuring that the composing logic gates retain their expected functionality when additional gates are included in the program. Engineered CRISPR/dCas9 systems are a promising tool to create large-scale transcriptional programs, given that one can design single guide RNAs (sgRNAs) to target dCas9 to any desired DNA sequence to perform transcriptional repression (CRISPRi) or activation (CRISPRa). This allows, in principle, an unlimited library of orthogonal parts. However, it has become apparent that increased numbers of sgRNAs in a circuit reduces each sgRNA’s ability to repress its targets, since sgRNAs load dCas9 such that when dCas9 is bound to one sgRNA it is not available for other sgRNAs. This loading phenomenon couples theoretically independent gates and results in severe performance degradation. To solve these problems, it is necessary to devise engineering approaches to mitigate effects of dCas9 loads. This project will design regulated dCas9 generators, wherein a feedback controller regulates unbound dCas9 level to a constant value, independent of the presence of gRNA loads. Repression circuits will be considered first and activation circuits along with mixed circuits will be addressed in a second step. The performance of the regulated dCas9 generators will be demonstrated on increasingly complex layered logic gates, including cascades and NAND gates. These regulated generators will ensure that any one gate’s functionality is preserved when additional gates are included in a system. Ultimately, the use of regulated dCas9 generators will enable increased scale, complexity, and reliability of CRISPR/dCas9 programs.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.