A Versatile Strategy for the Effective Multiplexing of Sgrnas for Synthetic Biology Applications

CRISPR/Cas9 based approaches have emerged as a key technology to address a wide range of basic biological questions and biotechnological challenges. In recent years applications have gone beyond genome editing to include implementations in the specific up- or down regulation of gene transcription programs or the visualisation and tracking of targeted gene loci in live cells. Many of these applications require the simultaneous expression of multiple guideRNAs in the same cell, yet the co-transfection of several sgRNA expressing plasmids can be problematic. We have devised a simple and versatile method for the production of highly multiplexed guideRNA expression arrays that can be efficiently integrated into a wide range of CRISPR application constructs.