In a recent communication in Nature Methods Kiani et al investigated the use of Cas9-VPR to serve simultaneously as an DNA endonuclease and a gene activator/repressor. That native Cas9 could be used for both purposes at the same time hinged on the observation that truncated guide RNAs (≤16 nt long) allow Cas9 to bind specific target sequences without cleaving the DNA.
The relatively easy means of programming the Cas9 protein has resulted in the use of Cas9 for multiple purposes including the generation of double and single-stranded DNA breaks, gene repression, and gene activation. These technologies require the use of various forms of Cas9, including the native and nickase forms, and an inactivated form, dCas9, which no longer possesses the ability to cleave DNA.
Kiani et al show that Cas9-VPR, which is an endonuclease-active Cas9 with the chimeric activation domain VPR (composed of the activation domains of VP64, P65 and Rta) can be used as a specific endonuclease, a transcriptional repressor and a transcriptional activator in the same cell.
Initially by targeting a transiently transfected fluorescent reporter in HEK293T cells, Kiani et al demonstrated that 20 or 18 nt guides resulted in cleavage of the targeted reporter, while 16 and 14 nt guides resulted in increased reporter expression comparable to dCas9-VPR with 20 nt guides. They were further able to reconstruct this pattern by targeting three endogenous genes, showing that Cas9-VPR with 14 nt long guides was comparable to dCas9-VPR with 20 nt long guides, with the added benefit that 14 nt long guides were less tolerant of mismatch, resulting in fewer off-targets and thus greater target specificity.
The ability to simultaneously utilize Cas9-VPR for DNA cleavage as well as gene activation/repression allows for a single Cas9 molecule to serve multiple functions for the generation of elegant gene circuits.
Kiani et al demonstrated this by designing Pol III driven 14 nt guides for enhanced expression of an EYFP marker and 20 nt guides targeting the CRISPR-activatable promoter (CAP) of the EYFP, where the 20 nt guides were expressed as an introns of an iRFP marker under control of a tet-on tetracycline response element promoter. Under this system, EYFP expression was activated by the Cas9-VPR-14 nt guide complex, and reduced when the Cas9-VPR-20 nt guide complex (the “kill switch”) was simultaneously present, due to the DNA cleavage of the CAP.
This work shows exciting possibilities of connecting the versatility of Cas9-VPR with existing genetic tools in insects to generate powerful and elegant gene circuits and switches.
Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJK, Ebrahamkhani MR, Collins JJ, Weiss R, Church G. 2015. Cas9 gRNA engineering for genome editing, activation and repression. Nature Methods; DOI:10.1038/NMETH.3580