Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells.
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.
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Lior
L
Nissim
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Samuel D
SD
Perli
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Alexandra
A
Fridkin
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Pablo
P
Perez-Pinera
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: timlu@mit.edu.
Timothy K
TK
Lu
D064113:CRISPR-Cas Systems; D005786:Gene Expression Regulation / Q000502:physiology; D053263:Gene Regulatory Networks; D057809:HEK293 Cells; D006801:Humans; D007438:Introns / Q000235:genetics / Q000502:physiology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism; D058615:Synthetic Biology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D058727:RNA, Small Untranslated
D035683:MicroRNAs; D016337:RNA, Catalytic; D014157:Transcription Factors; D012319:RNA Polymerase II; D058727:RNA, Small Untranslated