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

• Published in: G3 : genes - genomes - genetics 2023-04, Vol.13 (4)
• Main Authors: Warsinger-Pepe, Natalie, Chang, Carly, Desroberts, Connor R, Akbari, Omar S
• Format: Article
• Language: English
• Subjects: Animals; CRISPR-Cas Systems; Drosophila melanogaster - genetics; Drosophila Proteins - genetics; Heat-Shock Proteins - genetics; Hot Temperature; Investigation; Promoter Regions, Genetic; Response Elements; Temperature
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1093/g3journal/jkad024

Abstract 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.