Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

SUMMARYGenetic manipulation of mammalian cells is instrumental to modern biomedical research but is currently limited by poor capabilities of sequentially controlling multiple manipulations in cells. Currently, either highly multiplexed manipulations can be delivered to populations of cells all at one time, or gene regulatory sequences can be engineered to conditionally activate a few manipulations within individual cells. Here, we provide proof-of-principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 RNA-guided nuclease and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single guide RNA (sgRNA) - like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of genetic manipulations without the need for engineering cell type specific promoters or gene regulatory sequences. With the expanding diversity of available tools that utilize spCas9 to edit, repress or activate genes, this sgRNA-based system provides multiple levels for interfacing with host cell biology. In addition, ability of the system to progress through multiple modules from episomal plasmid DNA makes it suitable for applications sensitive to the presence of heterologous genomic DNA sequences and broadly applicable to biomedical research and mammalian cell engineering.Competing Interest StatementThe following competing interests are declared for RC, ART, HP, MSM, MR, and BJM as shareholders in Cellgorithmics, Inc., RC, HP, MSM, and BJM as cofounders of Cellgorithmics, Inc., and RC, HP, MSM, and BJM inventors on patent application number PCT/US2018/052211.View Full Text