RNA Engineering for CRISPR Synthetic Circuits and Endogenous Regulation
Various aspects and embodiments of the present disclosure relate to methods and compositions that combine multiple mammalian RNA regulatory strategies, including RNA triple helix structures, introns, microRNAs, and ribozymes with Cas-based CRISPR transcription factors and ribonuclease-based RNA processing in human cells. The methods and compositions of the present disclosure, in some embodiments, enable multiplexed production of proteins and multiple guide RNAs from a single compact RNA-polymerase-II expressed transcript for efficient modulation of synthetic constructs and endogenous human promoters.
The generation of robust, scalable gene networks that interfere minimally with endogenous cellular processes is fundamental to synthetic biology. The use of RNA regulatory strategies in combination with CRISPR/Cas transcription factors (CRISPR-TFs) allows for the conditional regulation of large-scale synthetic and endogenous gene networks in eukaryotic cells. The system is uniquely suited to build sophisticated circuits and transcriptional cascades that could be linked to specific events (e.g., cell cycle) or environments (e.g., chemical gradients).
Researchers
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methods and compositions for the production of guide rna
United States of America | Published application
Technology
Here we describe a genome editing toolkit for the generation of robust, scalable synthetic gene networks and native gene modulation in eukaryotic cells. Existing CRISPR/Cas methodology cannot be used for conditional gene regulation, as the guide RNAs (gRNAs) used to target DNA sites can only be expressed from RNA polymerase III (RNAP III) promoters, most of which are constitutively active. This technology produces gRNAs from RNA polymerase II (RNAP II) promoters, allowing for conditional gene expression and tunable responses to external inputs. The system integrates CRISPR-TFs with RNA regulatory machinery (e.g., Csy4 endonuclease, RNA triple helix) to produce functional gRNAs from RNAP II promoters while preserving downstream translation and expression of the harboring gene. The system is capable of multiplexed gRNA expression from one individual transcript, thus modulating multiple nodes from a single one and enabling sophisticated regulatory circuits with a large number of interconnections.
Problem Addressed
Current methods for genome editing (e.g., CRISPR/Cas) have the potential to modulate gene expression in a tunable manner but are often limited by a combination of scalability, lack of conditional regulation, and inefficient regulation of endogenous promoters. This genome engineering toolkit provides the means to regulate and synchronize multiplexed, large-scale synthetic gene networks in addition to modulating endogenous gene expression.
Advantages
- Multiplexed, large-scale genome engineering
- Synthesize multi-stage transcriptional cascades
- Regulate synthetic and endogenous networks
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