Imperial College Centre for Synthetic Biology, Imperial College London, London, SW7 2AZ, UK.
Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK.
Nat Commun. 2022 Aug 25;13(1):4984. doi: 10.1038/s41467-022-32603-7.
CRISPR gene activation and inhibition (CRISPRai) has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here we develop a method for near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems to increase CRISPRai multiplexing capacity and target gene flexibility. To achieve strong inducibility, we create a technology to silence gRNA expression within the array in the absence of the inducer, since we found that long gRNA arrays for CRISPRai can express themselves even without promoter. Using this method, we create a highly tuned and easy-to-use CRISPRai toolkit in the industrially relevant yeast, Saccharomyces cerevisiae, establishing the first system to combine simultaneous activation and repression, large multiplexing capacity, and inducibility. We demonstrate this toolkit by targeting 11 genes in central metabolism in a single transformation, achieving a 45-fold increase in succinic acid, which could be precisely controlled in an inducible manner. Our method offers a highly effective way to regulate genes and rewire metabolism in yeast, with principles of gRNA array construction and inducibility that should extend to other chassis organisms.
CRISPR 基因激活和抑制（CRISPRai）已成为一种强大的合成工具，可用于影响天然基因的表达，从而进行基础研究、细胞重编程和代谢工程。在这里，我们开发了一种方法，可近乎无泄漏地诱导表达多达 24 个来自两个正交的 CRISPR/Cas 系统的 gRNA 的多顺反子阵列，以增加 CRISPRai 的多重性和靶基因的灵活性。为了实现强诱导性，我们创建了一种在不存在诱导剂的情况下沉默阵列中 gRNA 表达的技术，因为我们发现，即使没有启动子，用于 CRISPRai 的长 gRNA 阵列也可以表达自己。使用这种方法，我们在工业相关酵母酿酒酵母中创建了一个高度调谐且易于使用的 CRISPRai 工具包，建立了第一个能够结合激活和抑制、大容量多重性和诱导性的系统。我们通过在单个转化中靶向中心代谢中的 11 个基因来证明该工具包的有效性，实现了琥珀酸的 45 倍增加，并且可以以诱导的方式进行精确控制。我们的方法为在酵母中调节基因和重新布线代谢提供了一种高效的方法，其 gRNA 阵列构建和诱导性的原则应该扩展到其他底盘生物。
