CRISPR-Cas systems are RNA-directed defense systems in prokaryotes that are revolutionizing genome engineering and gene regulation in bacteria through monkeys. While CRISPR-Cas systems are widely employed as heterologous tools, a number of industrially and medically relevant microorganisms naturally harbor these systems. This presents a unique opportunity to exploit endogenous CRISPR-Cas systems as convenient, ready-made tools in these microorganisms. Here, we demonstrate how to harness Type I CRISPR-Cas systems, the most common CRISPR-Cas systems in bacteria and in archaea, for programmable gene repression. Using the Type I-E CRISPR-Cas system native to Escherichia coli K-12 as a model, we report that this system could be readily converted into a programmable repressor through the deletion of the system’s cas3 endonuclease gene. Expressing designed CRISPR RNAs in the resulting strain instructed the modified system to tightly and specifically bind complementary DNA sequences. Targeting individual genes or their promoters led to potent and sequence-specific silencing of transcription. Furthermore, CRISPR RNAs could be encoded in arrays for multiplexed silencing and the generation of complex cellular phenotypes. We also establish simple design rules for tuning gene repression and demonstrate the system’s utility for performing combinatorial genetic screens and modulating pathway activity for dynamic metabolic control. Overall, this general strategy offers a convenient platform in synthetic biology for elucidating and engineering gene networks, particularly in the diversity of microorganisms that harbor these systems.

Endogenous CRISPR-Cas systems as convenient platforms for genetic screens and pathway engineering

249th National Meeting (2015)

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