Research Lab of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA.
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, Republic of Korea.
Nat Commun. 2021 May 11;12(1):2711. doi: 10.1038/s41467-021-22978-4.
Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.
时间调控多个基因的表达是复杂的生物学现象的基础。然而，用于顺序遗传扰动编程的可扩展和可推广的基因电路结构很少。在这里，我们描述了一种基于重组酶的模块化基因电路架构，由串联基因扰动盒（GPC）组成，通过仅用两种正交配体交替处理，可按定义的时间顺序顺序表达多个基因。我们使用串联 GPC 依次表达单引导 RNA，以编码转录级联反应，从而触发突变的顺序积累。我们构建了一个一体化的基因电路，可依次编辑基因组位点，在基因表达级联中的特定阶段同步细胞，并自行删除以确保安全。串联 GPC 为建模多阶段遗传变化（如肿瘤发生和细胞分化）提供了一种多层次的细胞编程工具。
