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

Lior Nissim, Samuel D. Perli, Alexandra Fridkin, Pablo Perez-Pinera, Timothy K. Lu
doi: https://doi.org/10.1101/004432
Lior Nissim
1Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
2Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA
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Samuel D. Perli
1Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
2Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA
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Alexandra Fridkin
1Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
2Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA
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Pablo Perez-Pinera
1Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
2Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA
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Timothy K. Lu
1Department of Biological Engineering and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
2Synthetic Biology Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA
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SUMMARY

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 of natural regulatory networks in human cells for basic biology, therapeutic, and synthetic-biology applications.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 23, 2014.
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An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells
Lior Nissim, Samuel D. Perli, Alexandra Fridkin, Pablo Perez-Pinera, Timothy K. Lu
bioRxiv 004432; doi: https://doi.org/10.1101/004432
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An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells
Lior Nissim, Samuel D. Perli, Alexandra Fridkin, Pablo Perez-Pinera, Timothy K. Lu
bioRxiv 004432; doi: https://doi.org/10.1101/004432

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