Header and Body 2

Problem Addressed

Gene regulatory networks are circuits in which molecular regulators, such as DNA-binding transcription factors, interact to control the expression of genes in a cell. Modulating gene regulatory networks, particularly through engineering of synthetic gene circuits, is essential for practical applications in biological engineering as well as for understanding the fundamentals of biological evolution. However, implementing synthetic biological circuits on the scale of hundreds of genes is difficult. As a result, CRISPR-based systems have emerged as useful tools for modulating regulatory circuits. Previous approaches utilize a catalytically inactive form of Cas9 (dCas9) that acts as a repressor after being directed to specific genes. However, when dCas9 is expressed at the high levels, as is common when building complex circuits, this protein becomes extremely toxic in bacterial cells due to non-specific binding. Therefore, there remains a need to engineer CRISPR/dCas9-based systems for regulatory network use in prokaryotes in which dCas9 can be expressed at high concentrations while maintaining specificity and avoiding toxicity.