CRISPR-Enabled Gene Regulation Tools for Biotechnology Applications

In this talk, I will introduce two new tools that we developed based on CRISPR-enabled gene regulation in bacteria, and one method based on P1 phagemid-enabled delivery of Cas9 nuclease. First, I will introduce a eukaryote-like bacterial CRISPRa system based on σ⁵⁴-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ⁵⁴-dependent promoters with biotechnology relevance in non-model bacteria, and act as a reusable scanning platform for readily optimizing metabolic pathways. Second, I will discuss the programmability of crRNA-tracrRNA hybridization which provides new sources of crRNAs and new biosensing applications for type II CRISPR systems. We show that how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro. Lastly, I will describe the use of a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. All these results demonstrate that CRISPR-enabled gene regulation is a powerful and versatile synthetic biology tool for diverse research and biotechnology applications.

Learning Objectives:

1. Design principles and biotech applications of a eukaryote-like CRISPR-enabled gene activation tool in bacteria.

2. Summarize principles and RNA sensing applications of reprogramming tracrRNAs in type II CRISPR system.

3. Design principles of engineering P1 phagemid to deliver CRISPR-Cas9 antimicrobials against Shigella flexneri, a dysentery-causing gut pathogen.