Abstract
The nuclease-deactivated variant of CRISPR-Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable orthologous dCas9 or Cpf1 effector proteins, thus opening options for multidimensional control of functional activities through combinations of orthogonal, drug-tunable artificial transcription factors.
Highlights
The nuclease-deactivated variant of CRISPR-Cas[9] proteins fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells
As the dCas[9] protein will readily combine with any supplied guide RNA in a cell, a clear drawback is that the combined use of different functionalities is troublesome and switching between different regulatory states, a highly desired requisite for the proper functioning of regulatory circuits, is precluded by the persistence of activating or repressing dCas[9]
The ecDHFR destabilising domain is an unfolded, structurally unstable domain derived from the Escherichia coli dihydrofolate reductase (DHFR) gene, which was evolved for enhanced instability by introduction of two additional missense mutations (R12Y/Y110I), which when fused to proteins of interest targets the fusion protein for rapid proteasomal degradation[12]
Summary
The nuclease-deactivated variant of CRISPR-Cas[9] proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas[9] effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. The small-molecule drug trimethoprim (TMP) can bind and stabilise the ecDDD in the fusion protein and prevent its proteosomal degradation in a concentration dependent manner[12]
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