A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling
- National Centre for Biological Sciences-TIFR, GKVK Campus, India
Figures
Figure 1
Interconversion of phosphoinositides in eukaryotic cells.
The metabolic pathway by which the different phosphorylated forms of phosphatidylinositol are interconverted is represented. The kinase and phosphatase reactions are indicated by green and black …
Figure 2
Generation of dgRNA targeting PI signaling genes.
(A) Workflow for the generation of dgRNA transgenic flies. For each of the 103 PI signaling genes in the Drosophila genome, two gRNAs were designed using flyCRISPR and verified using the CRISPR …
Figure 3
Design and validation of UAS-Cas9-T2A-eGFP.
(A) A schematic of the Cas9-T2A-eGFP transgene indicating the presence of a nuclear localization signal (NLS-blue box) at the N and C termini of Cas9; Cas9 is shaded in pink. An eGFP sequence …
Figure 4 with 2 supplements
A genetic screen employing the dgRNA transgenic fly library to identify PI signaling genes in eye development.
(A) Genomic PCR of CG5734 as a representative example to show that tissue specific gene deletion can be obtained by driving UAS-Cas9-T2A-eGFP specifically in the eyes using eyGal4 in the presence of …
Figure 4—figure supplement 1
Genomic deletion using Gal4/UAS system.
(A) Genomic PCR of Pi3K92E (Dp110) as an example to show that when comparatively larger genomic regions are deleted (in this case ~4 Kb- See Supplementary file 1), only the mutant band is amplified …
Figure 4—figure supplement 2
Genomic deletion obtained in CRISPR tissue-specific KOs is comparable irrespective of the presence or absence of phenotype.
Genomic PCR is done using heads of the flies from appropriate genotypes as indicated. dgRNA targeting synj and rdgB were chosen as examples of genes that did not show expected phenotypes, while …
Figure 5
Combinatorial use of dgRNAs to study genetic interactions regulating developmental processes.
(A) Knockdown of Dp110 results in smaller eyes compared to control eyes as seen with two different RNAi lines (Dp110RNAi #1: BDSC 61182 and Dp110RNAi #2: BDSC 27690). (B) CRISPR mediated Dp110 …
Figure 6
Generating germ line fly knockouts using dgRNA transgenics.
The PI transfer protein RDGB (retinal degeneration B) is a good example to demonstrate the need for whole fly knockouts. (A) Western blot analysis showed that eye-specific Cas9 expression was not …
Author response image 1
Verification of OCRL protein depletion when using CRISPR/Cas9 genome engineering reagents.
(A) Western blot from protein lysates of 3rd instar larvae from wild type and dOCRLKO. The dOCRL polypeptide of the expected Mr of ca. 100 kDa is completely absent in dOCRLKO lysates. Protein levels …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w1118 | Bloomington Drosophila Stock Center | BDSC 3605 | |
| Genetic reagent (D. melanogaster) | AB1-Gal4 | Bloomington Drosophila Stock Center | BDSC 1824 | y1 w*; P{GawB}AB1 |
| Genetic reagent (D. melanogaster) | UAS-Cas9 | Bloomington Drosophila Stock Center | BDSC 54592 | P{hsFLP}1, y1 w1118; P{UAS-Cas9.P}attP2/TM6B, Tb1 |
| Genetic reagent (D. melanogaster) | attP40 | Bloomington Drosophila Stock Center | BDSC 25709 | y1 v1 P{nos-phiC31\int.NLS}X; P{CaryP}attP40 |
| Genetic reagent (D. melanogaster) | attP2 | Bloomington Drosophila Stock Center | BDSC 25710 | P{nos-phiC31\int.NLS}X, y1 sc1 v1 sev21; P{CaryP}attP2 |
| Genetic reagent (D. melanogaster) | ey-Gal4 | Bloomington Drosophila Stock Center | BDSC 5534 | w*; P{GAL4-ey.H}3–8 |
| Genetic reagent (D. melanogaster) | rdgB9 | Bloomington Drosophila Stock Center | BDSC 27337 | |
| Genetic reagent (D. melanogaster) | nos-Cas9 | Bloomington Drosophila Stock Center | BDSC 54591 | y1 M{nos-Cas9.P}ZH-2A w* |
| Genetic reagent (D. melanogaster) | vasa-Cas9 (3) | Bloomington Drosophila Stock Center | BDSC 51324 | w1118; PBac{vas-Cas9}VK00027 |
| Genetic reagent (D. melanogaster) | vasa-Cas9 (1) | Bloomington Drosophila Stock Center | BDSC 51323 | y1 M{vas-Cas9}ZH-2A w1118/FM7c |
| Genetic reagent (D. melanogaster) | Act5c-Cas9 | Bloomington Drosophila Stock Center | BDSC 54590 | y1 M{Act5C-Cas9.P}ZH-2A w* |
| Genetic reagent (D. melanogaster) | nub-Gal4;UAS-Cas9 | Bloomington Drosophila Stock Center | BDSC 67086 | w*; P{GawB}nubbin-AC-62; P{UAS-Cas9.P2}attP2 |
| Genetic reagent (D. melanogaster) | PI3K RNAi #1 | Bloomington Drosophila Stock Center | BDSC 61182 | y1 sc* v1 sev21; P{TRiP.HMC05152}attP40 |
| Genetic reagent (D. melanogaster) | PI3K RNAi #2 | Bloomington Drosophila Stock Center | BDSC 27690 | y1 v1; P{TRiP.JF02770}attP2/TM3, Sb1 |
| Cell line (D. melanogaster) | S2 R+ | Drosophila Genomics Resource Center | Stock number 150 | |
| Antibody | Anti-Cas9 (Mouse monoclonal) | Takara | Cat#632628 (CloneTG8C1) | IF(1:200), WB (1:2000) |
| Antibody | Anti-OCRL (Rabbit polyclonal) | Gift from Avital Rodal | IF(1:50) | |
| Antibody | Anti-GFP (Rabbit polyclonal) | Abcam | Cat#: ab13970 | IF (1:5000) |
| Antibody | Anti-rdgB (Rat polyclonal) | Yadav et al., 2015 | WB (1:4000) | |
| Antibody | Anti-GFP (Mouse monoclonal) | Santacruz | Cat # SC 9996 | WB (1:2000) |
| Antibody | Anti-Syntaxin A1 (Mouse monoclonal) | DHSB | Cat # 8C3 | WB (1:1000) |
| Antibody | Anti-beta tubulin | DHSB | Cat # E7C | WB (1:4000) |
| Recombinant DNA reagent | pBS-hsp70-Cas9 (plasmid) | Addgene | Cat# 46294 | |
| Recombinant DNA reagent | pAC-Cas9-sgRNA (plasmid) | Addgene | Cat# 49330 | |
| Recombinant DNA reagent | pBFvU6.2 (plasmid) | NIG (Japan) | https://shigen.nig.ac.jp/fly/nigfly/cas9PlasmidsListAction.do | |
| Recombinant DNA reagent | pBFvU6.2B (plasmid) | NIG (Japan) | https://shigen.nig.ac.jp/fly/nigfly/cas9PlasmidsListAction.do | |
| Recombinant DNA reagent | pUASt (plasmid) | Brand and Perrimon, 1993 | ||
| Commercial assay or kit | Whole Genome DNA library prep kit | Illumina | FC-121–4002 | |
| Commercial assay or kit | Agilent high sensitivity DNA kit | Agilent | 5067–4626 | |
| Commercial assay or kit | Hiseq 2500 | Illumina | SY-401–2501 | |
| Software, algorithm | gRNA target finder | http://targetfinder.flycrispr.neuro.brown.edu/ | ||
| Software, algorithm | gRNA efficiency tool | https://www.flyrnai.org/evaluateCrispr/ | ||
| Other | DAPI stain | Invitrogen | D1306 | (1 µg/mL) |
Additional files
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Supplementary file 1
List of all 103 Drosophila PI-signaling genes against which dgRNAs have been generated.
The table indicates the CG numbers, gene names and what chromosome each of the genes are located on. For the phosphoinositide binding proteins, the various phosphoinositides either established or predicted to bind each protein have been listed. The table also has the closest human orthologs and associated human diseases from the Online Mendelian Inheritance in Man (OMIM) database indicated. The sequences of gRNA one and gRNA two for each gene are listed along with the size of the genomic deletion expected from these gRNA combinations, and the actual size of genomic deletion obtained in flies when crossed to Act5c-Cas9. The phenotypes obtained when these genes were deleted specifically in the eye and the efficiency (in terms of absolute numbers of flies with deletion as compared to total number of F2 flies) of whole fly gene knockout generation using either Vasa-Cas9, Act5c-Cas9 or Nos-Cas9 has been mentioned.
- https://cdn.elifesciences.org/articles/55793/elife-55793-supp1-v2.xlsx
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Supplementary file 2
List of all oligonucleotides used in this study is provided.
The sequence of each oligonucleotide is provided.
- https://cdn.elifesciences.org/articles/55793/elife-55793-supp2-v2.docx
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Supplementary file 3
Isogenized attP40 fly stock genomic sequence variants comparison with the reference genome.
The table shows chromosome location wise comparison between the isogenized attP40 stock with the reference genome. Average coverage for each sequence suggests the number of times each region has been sequenced. The frequency at which the variation is observed has been mentioned. A sequence quality of >Q30 score, which corresponds to >99.9% accuracy has been obtained. Each variation has been mapped based on genomic annotation of coding vs non-coding region. If variation in the coding region leads to amino-acid changes, it has been tabulated.
- https://cdn.elifesciences.org/articles/55793/elife-55793-supp3-v2.xlsx
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Supplementary file 4
S2R+ cell lines genomic sequence variants comparison with the reference genome.
The table shows chromosome location wise comparison between the S2R+ cells with the reference genome. Average coverage for each sequence suggests the number of times each region has been sequenced. The frequency at which the variation is observed has been mentioned. A sequence quality of >Q30 score, which corresponds to >99.9% accuracy has been obtained. Each variation has been mapped based on genomic annotation of coding vs non-coding region. If variation in the coding region leads to amino-acid changes, it has been tabulated.
- https://cdn.elifesciences.org/articles/55793/elife-55793-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55793/elife-55793-transrepform-v2.pdf
