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Abstract:
Certain cellular processes are dose-dependent, requiring specific quantities or stoichiometries of gene products, as exemplified by haploinsufficiency and sex-chromosome dosage compensation. Understanding dosage-sensitive processes requires tools to quantitatively modulate protein abundance. Here we present CasTuner, a CRISPR-based toolkit for analog tuning of endogenous gene expression. The system exploits Cas-derived repressors that are quantitatively tuned by ligand titration through a FKBP12F36V degron domain. CasTuner can be applied at the transcriptional or post-transcriptional level using a histone deacetylase (hHDAC4) fused to dCas9, or the RNA-targeting CasRx, respectively. We demonstrate analog tuning of gene expression homogeneously across cells in mouse and human cells, as opposed to KRAB-dependent CRISPR-interference systems, which exhibit digital repression. Finally, we quantify the system’s dynamics and use it to measure dose-response relationships of NANOG and OCT4 with their target genes and with the cellular phenotype. CasTuner thus provides an easy-to-implement tool to study dose-responsive processes in their physiological context.
