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

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Noviello,  Gemma
Systems Epigenetics (Edda G. Schulz), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Gjaltema,  Rutger A. F.
Systems Epigenetics (Edda G. Schulz), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Schulz,  Edda G.
Systems Epigenetics (Edda G. Schulz), Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Citation

Noviello, G., Gjaltema, R. A. F., & Schulz, E. G. (2022). CasTuner: a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression. bioRxiv, 511019. doi:10.1101/2022.10.05.511019.


Cite as: https://hdl.handle.net/21.11116/0000-000B-635A-1
Abstract
Certain cellular processes are dose-dependent, requiring a specific quantity of gene products or

a defined stoichiometry between them. This is exemplified by haploinsufficiency or by the need

for dosage compensation for X-linked genes between the sexes in many species.

Understanding dosage-sensitive processes requires the ability to perturb endogenous gene

products in a quantitative manner. Here we present CasTuner, a CRISPR-based toolkit that

allows analog tuning of endogenous gene expression. In the CasTuner system, activity of

Cas-derived repressors is controlled through a FKBP12F36V degron domain and can thereby be

quantitatively tuned by titrating the small molecule degrader dTAG-13. The toolkit can be applied

at the transcriptional level, using the histone deacetylase hHDAC4 fused to dCas9, or at the

post-transcriptional level, using the RNA-targeting CasRx. To optimise efficiency, inducibility and

homogeneity of repression we target a fluorescently tagged endogenous gene, Esrrb, in mouse

embryonic stem cells. Through flow cytometry, we show that CasTuner allows analog tuning of

the target gene in a homogeneous manner across cells, as opposed to the widely used KRAB

repressor domain, which exhibits a digital mode of action. We quantify repression and

derepression dynamics for CasTuner and use it to measure dose-response curves between the

pluripotency factor NANOG and several of its target genes, providing evidence for

target-specific dose dependencies. CasTuner thus provides an easy-to-implement tool to

perturb gene expression in an inducible, tunable and reversible manner and will be useful to

study dose-responsive processes within their physiological context