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POSTECH Life Science

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Research Highlight

Logical regulation of endogenous gene expression usingpr ogrammable, multi-input pr ocessing CRISPR guide RNAs

2024-07-01 171

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The CRISPR-Cas system provides a versatile RNA-guided approach for a broad range of applications. Thanks to advances in RNA synthetic biology, the engineering of guide RNAs (gRNAs) has enabled the conditional control of the CRISPR-Cas sy stem. Ho w e v er, achie ving precise regulation of the CRISPR-Cas sy stem f or efficient modulation of internal metabolic processes remains challenging. In this w ork, w e de v eloped a robust dCas9 regulator with engineered conditional gRNAs to enable tight control of endogenous genes. Our conditional gRNAs in Esc heric hia coli can control gene expression upon specific interaction with trigger RNAs with a dynamic range as high as 130-f old, e v aluating up to a three- input logic A OR (B AND C). The conditional gRNA-mediated targeting of endogenous metabolic genes, lacZ , malT and poxB , caused differential regulation of growth in Esc heric hia coli via metabolic flux control. Further, conditional gRNAs could regulate essential cytoskeleton genes, ftsZ and mreB , to control cell filamentation and division. Finally, three types of two-input logic gates could be applied for the conditional control of ftsZ regulation, resulting in morphological changes. The successful operation and application of conditional gRNAs based on programmable RNA interactions suggests that our system could be compatible with other Cas-effectors and implemented in other host organisms.

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