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Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster

Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for...

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Autores principales: Warsinger-Pepe, Natalie, Chang, Carly, Desroberts, Connor R, Akbari, Omar S
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085756/
https://www.ncbi.nlm.nih.gov/pubmed/36705519
http://dx.doi.org/10.1093/g3journal/jkad024
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author Warsinger-Pepe, Natalie
Chang, Carly
Desroberts, Connor R
Akbari, Omar S
author_facet Warsinger-Pepe, Natalie
Chang, Carly
Desroberts, Connor R
Akbari, Omar S
author_sort Warsinger-Pepe, Natalie
collection PubMed
description Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for both basic and applied studies, to precisely control gene expression. Here we characterize the use of Polycomb response elements and the inducible Heat-shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat-shock durations and rearing temperatures. We show that Polycomb response elements can significantly reduce expression of Cas9 under Heat-shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. We further demonstrate that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat-shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance.
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spelling pubmed-100857562023-04-12 Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster Warsinger-Pepe, Natalie Chang, Carly Desroberts, Connor R Akbari, Omar S G3 (Bethesda) Investigation Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for both basic and applied studies, to precisely control gene expression. Here we characterize the use of Polycomb response elements and the inducible Heat-shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat-shock durations and rearing temperatures. We show that Polycomb response elements can significantly reduce expression of Cas9 under Heat-shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. We further demonstrate that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat-shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance. Oxford University Press 2023-01-27 /pmc/articles/PMC10085756/ /pubmed/36705519 http://dx.doi.org/10.1093/g3journal/jkad024 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigation
Warsinger-Pepe, Natalie
Chang, Carly
Desroberts, Connor R
Akbari, Omar S
Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster
title Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster
title_full Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster
title_fullStr Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster
title_full_unstemmed Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster
title_short Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster
title_sort polycomb response elements reduce leaky expression of cas9 under temperature-inducible hsp70bb promoter in drosophila melanogaster
topic Investigation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085756/
https://www.ncbi.nlm.nih.gov/pubmed/36705519
http://dx.doi.org/10.1093/g3journal/jkad024
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