RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.
Bibliographical noteFunding Information:
We thank members of the Lu lab, especially Ramiz Daniel and Ky Lowenhaupt, for helpful discussions. We thank Lila Wroblewska for an mKate2 plasmid expressing intronic miRNA and Jeremy Wilusz and Courtney JnBaptiste for the cGFP _MALAT1_3′ plasmid. L.N. would like to thank Dr. Adina Binder-Nissim for support. This work was supported by the Defense Advanced Research Projects Agency and the National Institutes of Health (DP2 OD008435 and P50 GM098792).