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

Parts used

Key findings

• Placing bithoraxoid (bxd) Polycomb response element (PRE) repeats of about 2.5 kb and two gypsy insulator elements upstream of the Drosophila melanogaster Hsp70Bb promoter driving Cas9-T2A-eGFP (PRE-Hsp70BbCas9, integrated by ɸC31 at attP2) lowers Cas9 transcript levels in uninduced flies. By RT-qPCR in adult females without heat shock, Hsp70BbCas9 without PREs expresses 85.85 ± 2.63-fold more Cas9 RNA than PRE-Hsp70BbCas9 at 18°C rearing, 38.80 ± 12.11-fold more at 21°C and 7.41 ± 3.90-fold more at 26°C.
• PREs upstream of the Hsp70Bb promoter do not fully silence Cas9: PRE-Hsp70BbCas9 flies still carry Cas9 transcripts without heat shock, at 0.000047 ± 0.000017 (18°C), 0.000023 ± 0.0000015 (21°C) and 0.000034 ± 0.000014 (26°C) relative to RpL32. The difference in relative Cas9 transcript level between Hsp70BbCas9 and PRE-Hsp70BbCas9 without heat shock is significant at 18°C and 21°C rearing but not at 26°C, so higher rearing temperature appears to negate the PRE effect on uninduced Hsp70Bb promoter activity.
• A 37°C heat shock significantly raises Cas9 transcript levels in PRE-Hsp70BbCas9 flies. Relative to RpL32, Cas9 RNA at 18°C rearing is 0.0063 ± 0.0013 after 30 min, 0.0058 ± 0.00069 after 1 h and 0.0034 ± 0.00038 after 2 h, versus 0.000047 ± 0.000017 without heat shock, and it also rises significantly after 1 h and 2 h at both 21°C and 26°C rearing. Average Cas9 RNA after 2 h is lower than after 1 h at every rearing temperature, possibly from rapid suppression during the 30 min recovery.
• Rearing PRE-Hsp70BbCas9 flies at 18°C or 21°C with a 2 h or a 30 min heat shock respectively can both minimize uninduced Cas9 expression and maximize induced expression. The Cas9 expression ratio of Hsp70BbCas9 to PRE-Hsp70BbCas9 is 133.01 ± 47.77 after a 30 min heat shock at 18°C and peaks at 820.49 ± 90.54 after 30 min at 21°C, and at 21°C the ratio is significantly higher after 30 min, 1 h (165.54 ± 24.47) and 2 h (412.57 ± 82.94) than without heat shock (38.8 ± 12.11).
• Hsp70BbCas9 combined with sgRNA:w (guide RNA against white) produces white or variegated eyes in nearly all F1 trans-heterozygous flies regardless of heat-shock duration, rearing temperature or maternal versus paternal Cas9 inheritance. All conditions at 18°C and 26°C average 100% mutant phenotype and 21°C rearing never drops below 84%, a rate closer to constitutive vasaCas9 (100%) than to nosCas9 (5.71% of F1 females and 6.38% of F1 males with maternal Cas9, 0% with paternal Cas9).
• PREs upstream of Hsp70BbCas9 modestly but significantly lower the rate of white-eye phenotypes with sgRNA:w in most conditions, but the difference loses significance as heat-shock duration lengthens. After a 2 h heat shock, PRE+ and PRE- rates do not differ significantly for maternal Cas9 F1 females at 18°C (98.63 ± 2.19% versus 100 ± 5.08%) and 21°C (90.20 ± 3.80% versus 96.53 ± 5.39%), or for paternal Cas9 F1 females at 18°C and F1 males at 21°C (92.86 ± 2.90% versus 100 ± 2.10%).
• PREs upstream of Hsp70BbCas9 reduce the severity of the white mutant eye phenotype with sgRNA:w. sgRNA:w; Hsp70BbCas9 flies mostly show strong eye variegation regardless of parental Cas9 contribution, rearing temperature or heat-shock duration, whereas sgRNA:w; PRE-Hsp70BbCas9 flies show a range from no variegation (for example paternal Cas9, 21°C, no heat shock or 30 min heat shock) to variegation that increases with 1 h and 2 h heat shocks at every rearing temperature.
• The PRE-Hsp70BbCas9 transgene suppresses its own mini-white marker at 26°C rearing. sgRNA:w; PRE-Hsp70BbCas9 flies reared at 26°C, and trans-heterozygous males with maternally inherited PRE-Hsp70BbCas9, could not be reliably scored for white eye phenotype because the PRE-suppressed mini-white gives low-contrast variegation in a white mutant background.
• Visible white eye phenotypes in sgRNA:w; Hsp70BbCas9 flies correspond to Cas9-induced DNA mutations. Sanger sequencing of the mini-white transgene in individual trans-heterozygous flies with white mutant phenotypes shows mutations at the target site with both maternally and paternally inherited Hsp70BbCas9, so mutant phenotype rate serves as a proxy for Cas9 function.
• Heat-shock-induced Hsp70BbCas9 with the low-efficacy guide dgRNA:ey (targeting eyeless on the heterochromatic 4th chromosome) generates eyeless phenotypes in under 10% of F1 flies in every condition, comparable to constitutive nosCas9 (0%) and vasaCas9 controls. The highest rates are 4.62 ± 0.18% in F1 females with maternal Hsp70BbCas9 at 26°C after 2 h heat shock and 8.22 ± 0.23% in F1 males with paternal Hsp70BbCas9 after 2 h.
• PREs upstream of Hsp70BbCas9 further reduce the already rare eyeless phenotype generated with dgRNA:ey. PRE-Hsp70BbCas9 significantly lowers the mutant rate in every condition in which mutants occur, and eyeless mutants are found with PRE-Hsp70BbCas9 under only one condition, paternal Cas9 F1 females at 18°C rearing (1.39 ± 0.13%).
• Hsp70BbCas9 with the guide dgRNA:Ser (targeting Serrate) causes 100% pupal lethality in trans-heterozygous flies regardless of heat-shock duration or rearing temperature, and lethal pupae carry mutations at Ser gRNA target 1 or 2. Adding PREs upstream (PRE-Hsp70BbCas9) allows trans-heterozygous flies to survive and show visible serrated-wing Serrate phenotypes under most conditions, although why PREs rescue the lethality is not determined.

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.