Figures and data
cCCTomics Mediates Efficient Conditional Disruption of CCT Genes
(A) Schematic of cCCT gene span and principle of cCCTomics. A T2A-EGFP sequence was introduced at the 3’ end of CCT genes and their most or all coding regions (depending on attP-KO lines) were flanked by 34 bp FRT sequence. Both Flp-out (top) and GFP RNAi (down) could mediate CCT gene manipulation. (B-J) Expression of Trh (B-D), CCHa2 (E-G), and Dh31 (H-J) are efficiently disrupted by pan-neuronal expression of GFP-RNAi (B-D), pan-neuronal expression of Flp-out (E-G), and heatshock-Flp (H-J) respectively. Representative fluorescence images of R57C10-Gal4/+;TrhEGFP.FRT/+ (B), UAS-shRNAGFP/TrhEGFP.FRT (C), R57C10-Gal4/+; UAS-shRNAGFP/TrhEGFP.FRT (D), R57C10-Gal4/+;CCHa2EGFP.FRT/+; (E), UAS-Flp/CCHa2EGFP.FRT (F), R57C10-Gal4/+; UAS-Flp/CCHa2EGFP.FRT (G), Dh31EGFP.FRT with heatshock (H), hs-Flp/Dh31EGFP.FRT without heatshock (I), and hs-Flp/Dh31EGFP.FRT with heatshock are shown. Manipulation efficiency and experiment group fly number is noted on the right. Scale bar, 50um. (K-L) sleep profiles (K) and statistical analysis (L) of Flp-out induced nAChRβ2 neuronal knockout flies (dark red), nAChRβ2 knockout flies, and genotype controls (dark and blue). Sleep profiles are plotted in 30 min bins. In this and other figures, blank background indicates the light phase (ZT 0-12); shaded background indicates the dark phase (ZT 12-24). Daily sleep duration were significantly reduced in nAChRβ2 neuronal knockout files which is comparable to nAChRβ2 knockout. In all statistical panels, unless otherwise noted, 1) Numbers below each bar represent the number of flies tested. 2) Mean ± SEM is shown. 3) The Kruskal-Wallis test followed by Dunn’s post test was used. ***p<0.001. ** p<0.01. * p<0.05. n.s. p>0.05. Male flies were used unless otherwise noted.
C-cCCTomics Mediates Efficient Conditional Knockout of CCT Genes
(A) Schematic of C-cCCTomics principle. Cas9 and three sgRNAs are driven by GAL4/UAS system. Three tandem sgRNAs are segregated by fly tRNAGly and matured by RNase Z and RNase P. (B-G) Pan-neuronal knockout of Dh31 (D) and Pdf (G) by C-cCCTomics strategy. Representative fluorescence images presented expression of Dh31 (B-D) or anti-PDF (E-G). Pan-neuronal expression of Cas9 and sgRNA eliminated most (D) or all (G) fluorescent signal compared to control fly brains (B-C, E-F). Scale bar, 50 μm. (H) Activity profiles of pan-neuronal knockout of Pdfr and Pdfr-attpKO. Activity profiles were centered of the 12h darkness in all figures with evening activity on the left and morning activity on the right, which is different from general circadian literatures. Plotted in 30 min bins. (I) Statistical analysis of morning anticipation index (MAI), power, and period for pan-neuronal Pdf knockout and Pdfr-attpKO flies. Knocking out of Pdfr in neurons reduced both morning anticipation index, power and period significantly. (J-K) Statistical analysis of nAChRα2 (J) and nAChRβ2 (K) pan-neuronal knockout flies’ sleep phenotype. Sleep of these flies were not disrupted.
Efficiency Evaluation of Variations of Chromatin-Modulating Peptides Modified Cas9.
(A) Schematics of chromatin-modulating peptides modified Cas9. (B-J) Efficiency evaluation of Cas9 variants. Fluorescence imaging of R57C10-Gal4>UAS-sgRNAple (B), R57C10-Gal4>UAS-Cas9 (C-F), and R57C10-Gal4>UAS-Cas9, UAS-sgRNAple (G-J) flies are shown. Brains were stained with anti-TH (green). Scale bar is 50μm. (K) Anterior TH positive neurons numbers of (K-U). (L) Statistical analysis of ple knockout efficiency related to (K). Modified Cas9.M6 and Cas9.M9 showed an improved efficiency comparing to Cas9.HC. Student’s t test was used. (M) Statistical analysis of representational KO efficiency of Cas9 variants as related to Figure S5. Gene symbols on the right indicate tested genes. (N) Statistical analysis of Cas9 expression level. (O-P) Workflow of efficiency validation by next-generation sequencing (O) and Statistical analysis of single-site mutation ratios induced by Cas9 variants (P). Paired t test was used in (M), (N) and (P). (Q-R) Statistical analysis of sleep amount for nAChRα2 (Q) or nAChRβ2 (R) pan-neuronal knockout flies. Knockout of nAChRα2 and nAChRβ2 by modified Cas9.M9 significantly decreased flies’ sleep amount.
Genetic Dissection of Clk856 Labelled Clock Neurons.
(A-B) Schematic of intersection strategies used in Clk856 labelled clock neurons dissection, Flp-out strategy (A) and split-LexA strategy (B). The exact strategy used for each gene is annotated in Table S5. (C) Expression profiles of CCT genes in clock neurons. Gradient color denotes proportion of neurons that were positive for the CCT gene within each subset. The exact cell number for each subset is annotated in Table S4.
CNMa Regulation of Morning Anticipation in Clock Neuron
(A)Schematic of MAI, EAI, MAPI and EAPI definition. (B) Activity plots of male flies with CNMa knockout in clock neurons (red) and controls (blue, black and grey), plotted in 30 min bins. An advancement of morning activity peak was presented in CNMa clock-neuron-specific mutants (brown arrowhead). (C-E) Statistical analyses of MAPI, power, and period of flies in (B). MAPI was significantly increased in clock neurons-specific CNMa deficient flies (C) while power (D) and period (E) were not changed. (F) Schematic of CNMaKO generation. The entire encoding region of CNMa was replaced by an attP-SAstop-3P3-RFP-loxP cassette using CRISPR-Cas9 strategy. (G) Statistical analysis of MAPI of male CNMaKO flies (red) and controls (blue and black). MAPI significantly increased in male CNMaKO flies. (H) Activity plots of female CNMaKO flies (red) and controls (blue and black). (I) Statistical analysis of MAPI of female CNMaKO flies (red) and controls (blue and black). MAPI was significantly increased in female CNMaKO flies.
Expression, Projection and Trans-projection Feature of CNMa Neurons and Its Functional Subset
(A-C) Expression and projection patterns of CNMa-KI-Gal4 in the brain. Membrane, dendrites, and axon projections are labelled by mCD8::GFP (A), Denmark (B), and syt::eGFP (C) respectively. (D) Downstream neurons labelled through trans-tango driven by CNMa-KI-GAL4. Arrowheads indicate candidate downstream neurons: six neurons in PI, one pair in DN3, five pairs in LNd and about 15 pairs in SOG. (E-H) Intersection of DN1p CNMa neurons with DN1p labelled drivers. GMR51H05-GAL4 (E), GMR91F02-GAL4 (F), Pdfr-KI-GAL4 (G) and GMR79A11-GAL4 (H) were intersected with CNMa-p65AD, UAS-LexADBD, LexAop-myr::GFP. Two type I (E, G, H) neurons projected to anterior region and four type II (F) neurons had fewer projections to anterior region. Scale bar, 50μm. (I) MAPIs were significantly increased in all three DN1p drivers mediated CNMa knockout. (J-K) Activity plots of CNMa knockout in R51H05-GAL4 (J) and R91F02-GAL4 (N) neurons. R51H05-GAL4 mediated CNMa knockout flies showed an advanced morning activity peak (J), while R91F02-Gal4 mediated CNMa knockout flies did not (N). (Q-S) Statistical analyses of MAI, power and Period. Pdfr reintroduction in R79A11 and R51H05 neurons could partially rescue the MAI-decreased phenotype of Pdfr knockout flies. (T) Statistical analyses of MAI of Pdfr knocking out in R79A11-GAL4, CNMa-GAL4 and R51H05-GAL4 labelled neurons.
CNMaR Regulation of Morning Anticipation
(A) Schematic of CNMaRKO-p65AD generation. Most of the first exon in CNMaR was replaced by a T2A-p65AD-loxP-3P3-RFP-loxP cassette using CRISPR-Cas9 strategy and the T2A-p65AD was inserted in the reading frame of the remaining CNMaR codon. 3P3-RFP was removed latterly by Cre mediated recombination. (B-E) Activity plot (B, D) and statistical analysis (C, E) of male (B-C) or female (D-E) CNMaRKO-p65AD flies (red) and genotypical controls (blue and black). MAPI was significantly increased in both male and female CNMaRKO-p65AD flies. In this and other figures, “♀” denotes female flies. (F-H) Expression and projection patterns of CNMaR-KI-Gal4 in the brain. Scale bars, 50μm. (I-L) Activity plots (I, K) and statistical analyses (J, L) of CNMaR pan-neuronal knockout flies. Neuronal knockout of CNMaR increased MAPI (J, L).
Efficient Conditional Disruption of CCT Genes by cCCTomics
(A-I) Pan-neuronal knockdown of Dh31 (A-C), Mip (D-F) and Capa (G-I) by GFPi. All experimental fly brains (C, F, I) showed no GFP signal after knockdown by GFPi. As described in Fig 1, all percentages in the right represent gene disruption efficiency and n represent number of experimental flies. (J-U) Pan-neuronal knock out of Dh31, sNPF, Proc and SIFa by Flp-out strategy. No obvious GFP signal was found in the experimental fly brains (L, O, R), excepting one GFP positive neurons in SIFa flp-out group (U).
Gene Disruption of Target Genes by Induced shRNA
(A-J) Knockdown of Lk, Dh31 and sNPF by elav-Switch. Most GFP signal is lost in the experimental group (C, F, I).
Accurate Labeling of Target Genes by cCCT Lines
(A-J) Co-localization of fused EGFP labelled CCT genes and corresponding antibodies. All the EGFP labelled Lk (A, D) and Dsk (G) neurons (Green) were co-localized with anti-LK (B, E; merge C, F) and anti-DSK (H, merge I) signal (purple). Arrowheads represent specific anti-DSK signal (Wu et al., 2020).
Validation of Primary C-cCCTomics
(A) Gene span of Pdf. #1, #2 and #3 with red lines denote sgRNA targets regions in Pdf coding sequence. (B) DNA gel electrophoresis of PCR products after pdf KO by C-cCCTomics. “Ctrl” denotes PCR products from genomic DNA mixture of Act5C-GAL4/+; UAS-Cas9.P2/+ flies and UAS-sRNAPdf flies. “Test” denotes PCR products from Act5C-GAL4/ UAS-sRNAPdf; UAS-Cas9.P2 flies. (C-D) Sanger sequencing results of Ctrl (C) and Test (D) PCR products at #1 sgRNA target site. (E-H) Similar to (A-D) with Dh31 as target.
Efficiency validation by real-time quantitative PCR.
This figure corresponds to Fig 3M. The schematic at the top illustrates the principle of primer design. The gene symbol above each panel indicates the target of sgRNAs. The expression of the target gene in the experimental groups (where R57C10-GAL4 drives the expression of both Cas9 variants and sgRNA) was normalized to the control group (where R57C10-GAL4 drives sgRNA expression only).
Efficiency validation by high-throughput sequencing
This figure corresponds to Fig 3P. Gene symbol on the left side of each panel denotes the target gene, while the percentages on the right denotes mutation rate as calculated by CRISPResso2. A minimum of 10,000 reads were analyzed for each genotype.
Co-expression of Clk856 with CCT Genes
(01-24) Intersectional expression patterns of CCT drivers with Clk856. The exact strategy used for each gene is annotated in Table S5. Maximum neuron numbers were presented in each image. Note at the bottom right corner is category ID of CCT drivers in Rao Lab fly stock library. “f” denotes female fly brain, otherwise male brain is shown.
Co-expression of Clk856 with CCT Genes
(01-21) Intersectional expression patterns of CCT drivers with Clk856. The exact strategy used for each gene is annotated in Table S5. Maximum neuron numbers were presented in each image. Note at the bottom right corner is category ID of CCT drivers in Rao Lab fly stock library. “f” denotes female fly brain, otherwise male brain is shown.
Colocalization analyse of Dh31, Pdfr, and Pdf -KI-LexA with LNvs
LNvs are labels by anti-PDF. Yellow arrow heads indicate KI-LexA (Green, A, B) colocalized with anti-PDF (violet, A, B, C) and white arrow heads indicate KI-LexA did not colocalized with anti-PDF. (C) Pdf-KI-LexA labels all PDF positive LNvs but only scattered signal observed when intersect with CLK856-GAL4.
Disruption of CCT Genes due to Leaky Expression of Cas9.M9
(A) Activity plots of Pdfr or Pdf clock-neuron knockout flies (red) and control groups (blue, black, and gray). (B-D) Statistical analyses of morning anticipation index (B), EAPI (C) and power (D) after CCT genes were knockdown by Cas9.M9/sgRNAs. Leakage expression of Cas9.M9 and sgRNAs might disrupt target genes at certain levels (grey bar). Cas9.M9 denotes UAS-Cas9.M9. sgRNA denotes UAS-sgRNA.
Morning Activity Advanced by Loss of CNMa
(A) MAPI statistical analyses after mAChR-B, MsR1, nAChRα1 and CNMa knockout in clock neurons. Only the CNMa knockout in clock neurons increased MAPI significantly. (B-E) Activity plots (B, D) and statistical analyses (C, E) of male (B, C) and female (D, E) flies with CNMa knockout in clock neurons. Both male and female flies showed advanced morning activity patterns B, C) and increased MAPIs (D, E). UAS-sgRNACNMa/UAS-Cas9.M9 flies showed a slightly increased MAPI which indicate possible leakage expression. (F-G) Activity plots (F) and statistical analyses (G) of female flies with CNMa deficient in clock neurons with Cas9.M6 used.
Expression of CNMa and CNMaR
(A-B) Stinger::Red labeled neurons in brain driven by CNMa-KI-GAL4 (A) and CNMaR-KI-GAL4 (B)
Activity plots related to
(A-B) Activity plots of reintroducing Pdfr (A, related to Fig 6Q) or mutating Pdfr (B, related to Fig 6T) in DN1p subset neurons. 30 min per bin is plotted.
Impact on Viability of Cas9 Variants Expression by GMR57C10-GAL4
F1 ratio of UAS-Cas9 variants (male) cross GMR57C10-GAL4/cyo (virgin female). Expression of Cas9.M9 decreased progeny viability slightly.
** p<0.01. Two-student test was used.
cCCT Knockin fly list
cCCT knockin strategy can’t rescue all knockout phenotype
C-cCCTomics sgRNAs list
CCT gene expression profile of clock neuron
List of CCT genes intersected with Clk856 drivers
Phenotypes of CCT genes knocking out in clock neurons
arrhythmicity related to Figure 6R
Phenotypes of candidate CCT genes knockout in clock neurons
