Inducible and multiplex gene regulation using CRISPR–Cpf1-based transcription factors

Abstract

Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

“…The advantage of this set-up is that only the RuvC domain has to be inactivated to generate a catalytically inactive version of Cas12a (dCas12a). In the same study, it was also confirmed that a single crRNA array consisting of more than one crRNA is able to induce synergistic activation when targeted to the same promoter or multiplex activation when targeted to different promoters [86]. 2).…”

“…An interesting aspect of target site cleavage by Cas12a is that apparently, cleavage of the nontarget strand is essential for cleavage of the target strand, a contrast to Cas9-mediated cleavage [84,85]. In human cells it was reported that dCas12abased transcription factors can be used for transcriptional activation, through either regulation on the transcriptional or epigenetic level [86,87]. Like dCas9, dCas12a can be used as a platform to recruit different enzymes to sites of interest (Fig.…”

“…Currently, there are only few studies available showing the use of dCas12a as specific DNA binding protein. In human cells it was reported that dCas12abased transcription factors can be used for transcriptional activation, through either regulation on the transcriptional or epigenetic level [86,87]. By fusing a synthetic activator complex consisting of the VP16, p65, and Rta activator domains to dLbCas12a robust transcriptional activation could be achieved.…”

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…In the same study, it was demonstrated that transcriptional activation is inducible by drug treatment, using the split Lb‐dCas12a‐DmrA complex and a DmrC‐VPR or ‐p65 complex. Taking advantage that Cas12a can process its own crRNA in a multiplex single transcript, the same group confirmed the synergistic activation when targeting either the same or different promoters (Tak et al ). In Arabidopsis , dCas12a was already successfully applied as a transcriptional repressor fused with the SRDX repressor domain.…”

“…Instead of direct protein-linked mediated fusion of effectors to dCas9, factors can be indirectly bound to extended crRNAs (aptamers), via compatible peptide motifs. (Tak et al 2017). In Arabidopsis, dCas12a was already successfully applied as a transcriptional repressor fused with the SRDX repressor domain.…”