Chemical and optogenetic methods for post-translationally controlling protein function have enabled new discoveries and the engineering of synthetic cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of posttranslational behaviors that can be programmed we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs with engineered reader proteins that provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program diverse cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our receiver protein and suite Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

CRISPR/Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at offtarget and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the coadministration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating "scarless" editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a novel and flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites. The authors declare no competing financial interests.