An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells

BioRxiv : the Preprint Server for Biology
Lior NissimTimothy K Lu

Abstract

RNA-based regulation, such as RNA interference, and CRISPR/Cas transcription factors (CRISPR-TFs), can enable scalable synthetic gene circuits and the modulation of endogenous networks but have yet to be integrated together. Here, we combined multiple mammalian RNA regulatory strategies, including RNA triple helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing in human cells. We describe three complementary strategies for expressing functional gRNAs from transcripts generated by RNA polymerase II (RNAP II) promoters while allowing the harboring gene to be translated. These architectures enable the multiplexed expression of proteins and multiple gRNAs from a single compact transcript for efficient modulation of synthetic constructs and endogenous human promoters. We used these regulatory tools to implement tunable synthetic gene circuits, including multi-stage transcriptional cascades. Finally, we show that Csy4 can rewire regulatory connections in RNA-dependent gene circuits with multiple outputs and feedback loops to achieve complex functional behaviors. This multiplexable toolkit will be valuable for the construction of scalable gene circuits and the perturbation o...Continue Reading

Related Concepts

Genes
RNA
Transcription, Genetic
Catalytic RNA
RNA, Guide
Promoter
Hepatocyte Nuclear Factor 1-beta
Strategy
Human Cell Line
MicroRNAs

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