Fluorescent Guide RNAs Facilitate Development of Layered Pol II-Driven CRISPR Circuits

ACS Synthetic Biology
David J MennXiao Wang

Abstract

Efficient clustered regularly interspaced short palindromic repeat (CRISPR) guide RNA (gRNA) expression from RNA Polymerase II (Pol II) promoters will aid in construction of complex CRISPR-based synthetic gene networks. Yet, we require tools to properly visualize gRNA directly to quantitatively study the corresponding network behavior. To address this need, we employed a fluorescent gRNA (fgRNA) to visualize synthetic CRISPR network dynamics without affecting gRNA functionality. We show that studying gRNA dynamics directly enables circuit modification and improvement of network function in Pol II-driven CRISPR circuits. This approach generates information necessary for optimizing the overall function of these networks and provides insight into the hurdles remaining in Pol II-regulated gRNA expression.

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Citations

Sep 24, 2020·Biochemical Society Transactions·Javier Santos-Moreno, Yolanda Schaerli
Jun 4, 2020·Nature Communications·Javier Santos-MorenoYolanda Schaerli
Aug 21, 2021·Scientific Reports·Dashan Sun

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