Multiplexed and Programmable Regulation of Gene Networks with an

Integrated RNA and Crispr/Cas Toolkit in Human Cells

Lior Nissim1#, Samuel D. Perli2# Alexandra Fridkin1, Pablo Perez-Pinera1 and Timothy K.

Lu3,

(#) These authors contributed equally

(1) MIT, Cambridge, MA,

(2) EECS, MIT, Cambridge, MA,

(3) EECS/Biological Engineering, MIT, Cambridge, MA

CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to enable multiplexed modulation of synthetic and endogenous gene networks in a programmable fashion. However, gRNAs have so far have been expressed only from RNA polymerase III promoters, limiting the integration of CRISPR-TFs with mammalian RNA regulatory architectures. We present multiple strategies for the production of gRNAs in human cells from endogenous, RNA polymerase II promoters by employing Cas6/Csy4 based RNA processing, RNA-triple-helix structures, introns and ribozymes. In addition to generating multiple gRNAs from single transcripts and implementing multi-stage cascades, we integrate CRISPR- TFs with microRNAs to build RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. Our toolkit enables the design of scalable gene circuits and the modulation of endogenous regulatory networks for biology, therapeutic, and synthetic biology applications.