Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression

Abstract

CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing and transcriptional regulation. Because these RNA-directed immune systems are found in most prokaryotes, an opportunity exists to harness the endogenous systems as convenient tools in these organisms. Here, we report that the Type I-E CRISPR-Cas system in Escherichia coli can be co-opted for programmable transcriptional repression. We found that deletion of the signature cas3 gene converted this immune system into a programmable gene regulator capable of reversible gene silencing of heterologous and endogenous genes. Targeting promoter regions yielded the strongest repression, whereas targeting coding regions showed consistent strand bias. Furthermore, multi-targeting CRISPR arrays could generate complex phenotypes. This strategy offers a simple approach to convert many endogenous Type I systems into transcriptional regulators, thereby expanding the available toolkit for CRISPR-mediated genetic control while creating new opportunities for genome-wide screens and pathway engineering.

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

“…Studies have revealed that among Cas proteins in Type I CRISPR-Cas systems, Cas3 has as a key role in programing gene expression [30] by acting as a single-strand DNA nuclease and ATP-dependent helicase [31]. Csy1-4 proteins constitute the cascade [32], wherein Csy4 func-tions in processing crRNA transcripts, whereas Csy1-3 proteins are required to stabilize the crRNA produced by Csy4 [32].…”

A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future

“…In addition, the emergence of sgRNA design rules is promising to accelerate the development of CRISPRi even more [29]. Recent work is also showing that other CRISPR/Cas systems can be engineered, such as the endogenous type I-E system of E. coli [30 ], to create a diverse range of CRIPSR/Cas-based regulators, each with unique design principles and advantages.…”

Harnessing CRISPR–Cas systems for bacterial genome editing

“…Exploitation of endogenous and orthogonal systems CRISPR-Cas systems are found in approximately 46% of bacteria and 84% of Archaea [43], highlighting the potential for genome editing applications using endogenously active systems [44]. However, there is a general paucity of systems with characterized PAMs and confirmed activity in interference [45].…”