Engineering novel CRISPR-enabled gene regulatory devices in bacteria
Item statusRestricted Access
Embargo end date31/07/2022
CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive immune system of prokaryotes. Due to its DNA/RNA recognition ability mediated by nucleic acid complementary pairing, the CRISPR systems can be easily reprogrammed for various purposes. Today, CRISPR tools have been widely used in genome editing, gene regulation, molecular markers, biosensors, and related fields. However, the existing CRISPR tools are not sufficient to meet diverse needs. The development of novel CRISPR tools and the exploration of its programmability are vital to meet the needs of various applications while deepening our understanding of the CRISPR system. Here, we achieved a eukaryote-like CRISPR activation (CRISPRa) device in bacteria, which fills the gap for the programmable activation technology of σ54-dependent promoters. The unique mechanism of this CRISPRa provides several desired properties, enabling the design of complex gene circuits. I demonstrated new applications of this novel tool in both foundational research and industrial biotechnology. For supporting foundational research, I engineered this CRISPRa device as a platform to explore the programmability of crRNA-tracrRNA pairing. Thus, I found that the reprogrammed tracrRNAs can repurpose non-crRNAs such as mRNAs to crRNAs and trigger CRISPR regulation. For enabling industrial applications, I developed a reusable projection system of multi-gene expression profiles, which can separate the diversity of multi-gene expression profiles from specific circuits and store it in a universal library of multi-sgRNA generators. Further, I demonstrated that this tool enables high-quality, low-cost multi-gene expression profile screening. I hope that my efforts in the development and application of novel CRISPR regulatory devices will provide valuable inspiration to the CRISPR research field.