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 nuclease 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.

Figure 1. Targeted human endogenous gene regulation using individual crRNAs with dLbCpf1-based activators

(a) Activities of dLbCpf1-p65 or dLbCpf1-VPR using single crRNAs at three endogenous human genes (HBB, AR, and NPY1R) in HEK293 cells. Relative mRNA expression is calculated by comparison to the control sample in which no crRNA is expressed. *, statistically significant difference compared to the control sample by Student t-test (two-tailed test assuming equal variance, p < 0.05). (b) Schematic illustrating drug-dependent bi-partite dLbCpf1-based activator fusion proteins. (c) Activities of drug-dependent bi-partite dLbCpf1-based activators using single crRNAs at three endogenous human genes (HBB, AR, and NPY1R) in HEK293 cells. The crRNA used for each promoters was the one that showed the highest activity in (a) above. Data shown in (a) and (c) represent three biological independent replicates.

Figure 2. Multiplex and synergistic regulation of endogenous human genes by dLbCpf1-based activators

(a) Schematic illustrating multiplex expression of three crRNAs each targeted to the same endogenous gene promoter in the same cell. (b) Activities of dLbCpf1-DmrA(x4) and DmrC-VPR fusions (pink bars) or of dLbCpf1-DmrA(x4) and DmrC-p65 fusions (blue bars) with three single crRNAs, pooled sets of single crRNAs, and a MST encoding all three crRNAs on the HBB, AR, or NPY1R endogenous gene promoters. Synergistic effects of dLbCpf1-VPR/p65 fusions with MST crRNAs or individual pooled crRNAs are all statistically significant (Student t-test, two-tailed test assuming equal variance, p < 0.05) except for cases where n.s. (not significant) is indicated. (c) Schematic illustrating multiplex expression of three crRNAs each targeted to a different endogenous gene promoter in a single cell. (d) Simultaneous activation of three endogenous human genes using crRNAs expressed singly or from a MST together with dLbCpf1-VPR direct fusions (left panel), dLbCpf1-DmrA(x4) and DmrC-p65 fusions (middle panel), or dLbCpf1-DmrA(x4) and DmrC-VPR fusions (right panel). For the direct fusion experiment, a total of 250 ng of plasmids encoding the indicated crRNAs was used. For each drug-regulated fusion experiment, a total of 400ng of plasmids encoding the indicated crRNAs was used. Transcripts were measured in HEK293 cells using RT-qPCR with relative mRNA expression calculated by comparison to the control sample in which no crRNA is expressed. Representative data shown in (b) and (d) are of three biological independent replicates. hU6, human U6 Polymerase III promoter; DR; direct repeat sequence; n.s., not significant by Student t-test (p > 0.05); *, statistically significant by Student t-test (two-tailed test assuming equal variance, p< 0.05)