CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression
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...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-06-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-38909-4 |
| _version_ | 1797811447807344640 |
|---|---|
| author | Gemma Noviello Rutger A. F. Gjaltema Edda G. Schulz |
| author_facet | Gemma Noviello Rutger A. F. Gjaltema Edda G. Schulz |
| author_sort | Gemma Noviello |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T07:22:49Z |
| format | Article |
| id | doaj.art-f95255125c19470ba443765c7b518f68 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-13T07:22:49Z |
| publishDate | 2023-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-f95255125c19470ba443765c7b518f682023-06-04T11:33:13ZengNature PortfolioNature Communications2041-17232023-06-0114111710.1038/s41467-023-38909-4CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expressionGemma Noviello0Rutger A. F. Gjaltema1Edda G. Schulz2Systems Epigenetics, Otto Warburg Laboratories, Max Planck Institute for Molecular GeneticsSystems Epigenetics, Otto Warburg Laboratories, Max Planck Institute for Molecular GeneticsSystems Epigenetics, Otto Warburg Laboratories, Max Planck Institute for Molecular GeneticsAbstract 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.https://doi.org/10.1038/s41467-023-38909-4 |
| spellingShingle | Gemma Noviello Rutger A. F. Gjaltema Edda G. Schulz CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression Nature Communications |
| title | CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression |
| title_full | CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression |
| title_fullStr | CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression |
| title_full_unstemmed | CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression |
| title_short | CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression |
| title_sort | castuner is a degron and crispr cas based toolkit for analog tuning of endogenous gene expression |
| url | https://doi.org/10.1038/s41467-023-38909-4 |
| work_keys_str_mv | AT gemmanoviello castunerisadegronandcrisprcasbasedtoolkitforanalogtuningofendogenousgeneexpression AT rutgerafgjaltema castunerisadegronandcrisprcasbasedtoolkitforanalogtuningofendogenousgeneexpression AT eddagschulz castunerisadegronandcrisprcasbasedtoolkitforanalogtuningofendogenousgeneexpression |