ATFs are valuable tools for studying gene functions and transcriptional networks. Zinc-fingers and TALE transcription factors have been developed over the recent decades and show promises in both bioengineering and therapeutic applications3,9,10
. Here we established CRISPR-on as a novel class of artificial transcription factors based on the CRISPR/Cas system. The major advantage of this system is that only one dCas9 activator is required to activate multiple genes individually or simultaneously and that its DNA binding specificity is determined by sgRNAs, which are designed based on simple RNA/DNA complementarity.
Using CRISPR-on, we demonstrate robust activation of exogenous reporter genes in both human and mouse transformed cells as well as in ES cells. When the system was introduced into one-cell mouse embryos, efficient reporter gene activation was observed, raising the possi-bility of manipulating transcriptional networks in early embryos.
We achieved robust endogenous gene activation using the stronger activation domain VP160. Further optimization of activation domains, such as using different linker sequences, may improve the CRISPR-on activation efficiency even further. The promoter scanning experiments demonstrated that efficient activation of endogenous genes could be achieved by three to five sgRNAs binding within 300 bp region upstream of TSS. Using additional sgRNAs targeting further upstream or downstream regions did not significantly improve the level of induction. Our data suggest that only a small number of sgRNAs targeting the proximal promoter are sufficient to activate endogenous genes. While our paper was under review, similar results were reported showing synergistic and robust activation of endogenous genes by proximal binding of dCas9 activators15
We show here that the CRISPR-on system can be used for the simultaneous induction of at least three different endogenous genes. More significantly, we demonstrated that the stoichiometry of gene induction of multiple genes can be tuned by adjusting the relative amount of their cognate sgRNAs. Simultaneous activation of multiple endogenous genes with defined stoichiometry opens up novel opportunities for systems biology as it allows for the predictable manipulation of transcriptional networks.
Finally, with the ease of design and synthesis, a library of sgRNAs could be generated. When introduced into a cell line constitutively expressing dCas9 activator, gene activation screens mediated by RNA (RNAa) could be achieved. As the specificity components (sgRNA) can be separately designed and constructed from the effector component (Cas fusion proteins), the same library of sgRNAs could be used with different dCas9 fusions (e.g., VP160 domain for transactivation, KRAB domain for transcriptional repression, chromatin modifier domains for specific histone modification) to exert different functions at particular genomic loci.