Hanewich-Hollatz Mikhail H, Chen Zhewei, Hochrein Lisa M, Huang Jining, Pierce Niles A
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
Division of Engineering & Applied Science, California Institute of Technology, Pasadena, California 91125, United States.
ACS Cent Sci. 2019 Jul 24;5(7):1241-1249. doi: 10.1021/acscentsci.9b00340. Epub 2019 Jun 4.
A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.
引导RNA（gRNA）将CRISPR蛋白效应物的功能导向选定的靶基因，为构建多种模式的合成调控（编辑、沉默、诱导、结合）提供了一个通用的可编程平台。然而，gRNA持续活跃这一事实限制了将gRNA活性限制在期望的位置和时间的能力。为了实现对gRNA活性范围的可编程控制，我们在此应用动态RNA纳米技术的原理来构建条件引导RNA（cgRNA），其活性取决于RNA触发物的存在与否。这些cgRNA在两个层面上是可编程的，触发物结合序列控制效应物活性的范围，而靶标结合序列决定效应物活性的对象。我们展示了由RNA触发物条件性失活的持续活跃cgRNA（开→关逻辑）和由RNA触发物条件性激活的持续无活性cgRNA（关→开逻辑）的分子机制。对于每种机制，使用NUPACK中的反应途径设计器进行自动化序列设计，以设计一个由三个响应不同RNA触发物的cgRNA组成的正交文库。在表达cgRNA、触发物以及作为蛋白效应物的沉默dCas9时，对于“终止子开关”这种开→关机制，我们观察到的中位条件响应约为4倍；对于“夹板开关”这种开→关机制，约为15倍；对于“趾状开关”这种关→开机制，约为3倍；三种机制中每个cgRNA/触发物文库内的中位串扰分别为<2%、≈2%和约20%。为了测试在细菌中构建的cgRNA机制对哺乳动物细胞的适用性，以及测试其对不同效应物功能的通用性，我们在表达诱导性dCas9作为蛋白效应物的HEK 293T细胞中实现了终止子开关，观察到三个正交cgRNA/触发物对的中位开→关条件响应约为4倍，中位串扰约为30%。通过对蛋白效应物功能的范围和靶标提供可编程控制，cgRNA调节器为合成生物学提供了一个有前景的平台。
