CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression

Gemma Noviello, Rutger A. F. Gjaltema and Edda G. Schulz ()
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Gemma Noviello: Max Planck Institute for Molecular Genetics
Rutger A. F. Gjaltema: Max Planck Institute for Molecular Genetics
Edda G. Schulz: Max Planck Institute for Molecular Genetics

Nature Communications, 2023, vol. 14, issue 1, 1-17

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-38909-4

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