Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Genes targeted in vivo by dCas9-effectors in this study.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: In Vivo, Binding Assay

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Rb paralogs have similar transcriptional effects on E2F2 and Mpp6 reporters in S2 cells. a) For all experiments described here, S2 cells were transfected with actin -GAL4, one of the dCas9 effectors, a single gRNA, and a luciferase reporter. b) Schematic of the Mpp6 -luciferase reporter that was designed to be regulated by the Mpp6 promoter. gRNAs are identical to those used in vivo, but lack gRNA 6 and 7, as they bind within the protein coding sequence not present in the reporter. c) dCas9-Rbf1 recruitment to the Mpp6 reporter significantly represses luciferase expression from position 2 and 3 (50%). Position 5 repression appears to represent a nonspecific steric hindrance, as it is seen with dCas9 alone e). d) dCas9-Rbf2 has a similar pattern and degree of repression of this reporter as dCas9-Rbf1. e) dCas9 has little effect on this promoter aside from position 5, which suggests steric hindrance from downstream of the TSS. f) Schematic of the E2F2 -luciferase reporter that was designed to be regulated by the E2F2 promoter. Here, the gRNAs are in opposite orientation from what is shown in a), as this is the same genomic region, but on the opposite strand. g) dCas9-Rbf1 recruitment to the E2F2 reporter significantly represses luciferase expression from position A (25%) and position B (60%). Recruitment to position 2 causes the same level of repression as dCas9 alone, suggesting steric hindrance from this site, as seen in i). h) dCas9-Rbf2 has a similar pattern and degree of repression of this reporter as dCas9-Rbf1. i) dCas9 has little effect on this promoter, aside from position 2 and 1, which suggests that the E2F2 promoter is more sensitive than the Mpp6 promoter to recruitment of dCas9. Error bars indicate SEM, and * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Expression is normalized to the empty gRNA vector control.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Transfection, Luciferase, In Vivo, Sequencing, Expressing, Plasmid Preparation, Control

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Creation of an in vivo CRISPRi system in Drosophila to target Rb paralogs to endogenous gene promoters. a) The fly Rbf1 and Rbf2 FLAG-tagged coding sequences were fused to the C-terminus of the S. pyogenes nuclease dead Cas9 (dCas9; asterisks denote inactivating mutations), and placed under UAS expression. FLAG-tagged dCas9 was created as a negative control. dCas9-VPR was obtained as a fly line (#67055 from BDSC); VPR is a tripartite activator that was previously characterized as a Drosophila transcriptional activator ( Lin et al . 2015 ). b) Here, we generated homozygous flies expressing UAS:dCas9-Rb chimeras under the expression of nubbin -GAL4 (expression predominantly in the wing pouch of L3 wing discs; ; ). We crossed these fly lines to homozygous flies expressing 2 gRNAs for a single gene's promoter (from the DRSC). These tandem gRNAs bind within 400 bp of a gene's TSS, and are ubiquitously expressed. c) Progeny from the cross indicated in b) express 3 transgenes in order to target one of the dCas9 chimeras to a single gene's promoter in the L3 wing disc; these flies were used for most of the in vivo experiments described in this study. d) Generation of flies expressing UAS:dCas9-Rb effectors in the follicle cells of the adult female ovary using a traffic jam -GAL4 driver (#104055 from the DGRC). Fly crossing scheme is as depicted in b).

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: In Vivo, Expressing, Negative Control, Generated

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Targeting dCas9-Rb chimeras to endogenous promoters leads to gene-specific effects in the wing. a–c) Wings ( n = 100) were dissected from adult flies for phenotype analysis after dCas9-effectors were targeted to endogenous gene promoters in L3 wing discs. Representative images from each cross are shown. a) A non-targeting gRNA control (QUAS) demonstrates that expression of effectors not targeted to any locus on the genome do not cause adult wing phenotypes. b) Targeting the wg promoter with VPR leads to severe developmental defects, but Rb effects on this promoter are mild to non-existent. c) Targeting the E2F2 promoter with Rbf1 and Rbf2 leads to severe morphological defects that are Rb-specific, confirming the feasibility of the dCas9-Rb fusions in vivo in Drosophila. d–f) Quantification of the wings analyzed in a–c). Values are indicated as a proportion of the total number of wings. d) Quantification of images in a). Background effects are essentially non-existent when expressing the dCas9-effectors with a non-targeting gRNA. e) Quantification of images from crosses depicted in b). dCas9-VPR has a severe phenotypic effect on wg , due to wg overexpression, as previously reported ( Ewen-Campen et al . 2017 ). Minor phenotypes are observed in ∼20% of wings with dCas9-Rbf2. f) Quantification of images from crosses depicted in c). Effects of targeting the E2F2 promoter are severe with both Rb paralogs, and the phenotype is fully penetrant. Legend in f) is shown for all graphs.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Control, Expressing, In Vivo, Over Expression

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Rbf1 and Rbf2 recruitment to the E2F2/ Mpp6 promoter leads to Rb-specific repression. a) Diagram of the E2F2 gene, which shares its promoter with Mpp6 as a divergently paired gene. gRNA binding sites on the promoter of E2F2 , which overlaps with the promoter of Mpp6 . Horizontal lines below the genes are the locations of the E2F2 gRNAs, with approximate distance from the indicated E2F2 TSS. b, c) Expression level of E2F2 and Mpp6 measured in L3 wing discs after dCas9-effector recruitment, using RT-qPCR. Control samples are dCas9 flies crossed to a non-targeting gRNA fly line (QUAS). b) Rbf1 and Rbf2 significantly repress E2F2 expression. dCas9 alone leads to some modest repression, while the VPR activator has no significant effect on expression of E2F2 or Mpp6 . c) Rbf1 and Rbf2 significantly repress Mpp6 expression, to a greater degree than observed for E2F2 . Effects are significantly greater than by dCas9 alone. d, e) Expression level of E2F2 and Mpp6 measured in ovaries after dCas9-effector recruitment, using RT-qPCR. d) E2F2 is significantly repressed by dCas9-Rbf1, which is greater than dCas9-Rbf2 and dCas9 effects. e) Mpp6 is significantly repressed by dCas9-Rbf1, and dCas9-Rbf1 appears to be a better repressor than Rbf2, although the difference is not statistically significant. For b–e), values in the bars indicate the average expression, error bars are SEM, and asterisks denote statistically significant differences: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Control

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Position-sensitive Rb paralog effects on the E2F2/ Mpp6 bidirectional promoter. a) Schematic of single gRNAs targeting the 1 kb promoter between the E2F2 and Mpp6 transcribed regions. gRNA 4 is what is indicated as −577 in , and gRNA 5 is what is indicated as −672. 4 + 5 are the 2 tandem gRNAs used in . dCas9 effectors were recruited to these sites using one gRNA at a time. b) Adult wings ( n = 50) were dissected after targeting by dCas9, dCas9-Rbf1, and dCas9-Rbf2. Images are representative wings. The severe morphological defect which is clear with 4 + 5 is only observed with gRNA 4 or 5 alone with Rbf2. Rbf1 targeting with the single gRNA 4 or gRNA 5 does not produce this severe effect. c) Quantification of the percentage of wings with particular phenotypes as indicated in the legend on the bottom right. Milder effects were observed from most sites, with supernumerary bristles or ectopic veins forming in a small proportion of wings.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques:

Journal: G3: Genes | Genomes | Genetics

Article Title: Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

doi: 10.1093/g3journal/jkae238

Figure Lengend Snippet: Repression of E2F2 and Mpp6 in a distance-dependent manner. a) Schematic of gRNAs designed here to target the E2F2/ Mpp6 promoter. dCas9 effectors were recruited to these sites using one gRNA at a time, and L3 wing discs were dissected for RT-qPCR analysis. b) Expression of E2F2 after recruitment of dCas9-Rbf1 to each gRNA site. Position 2 is highlighted, where the greatest repression was measured (∼40%). c) Expression of Mpp6 after recruitment of dCas9-Rbf1 to each gRNA site. Positions 4 and 5 are highlighted, where the greatest repression was measured. Rbf1 is a better repressor when 2 molecules are recruited to the Mpp6 TSS. d) Expression of E2F2 after recruitment of dCas9-Rbf2 to each gRNA site. Position 2 is highlighted, where the greatest repression was measured (∼40%), identical to what was measured by dCas9-Rbf1. e) Expression of Mpp6 after recruitment of dCas9-Rbf2 to each gRNA site. Positions 4 and 5 are highlighted, where the greatest repression was measured. Whether a single molecule or 2 molecules of Rbf2 are recruited does not make a big difference in magnitude of repression (∼50%). f) Expression of E2F2 after recruitment of dCas9 to each gRNA site. dCas9 is not able to repress this gene from most sites. g) Expression of Mpp6 after recruitment of dCas9 to each gRNA site. dCas9 is not able to repress this gene from most sites, aside from a few. Error bars indicate SEM, and * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Full length dCas9 (with D10A, H840A substitutions) was obtained from the pcDNA-dCas9 vector (gift from Charles Gersbach; Addgene plasmid #47106; Perez-Pinera et al . 2013 ).

Techniques: Quantitative RT-PCR, Expressing