A pilot study for whole proteome tagging in Caenorhabditis elegans
- Columbia University, Department of Biological Sciences, Howard Hughes Medical Institute, United States
Figures
Figure 1
Pipeline for triple pooled CRISPR-Cas9-mediated gene tagging.
(A) Schematic of injection and screening strategies. FP, fluorescent protein. (B) Excitation and emission spectra of selected fluorescent proteins used in the pilot study. Theoretical brightness of the fluorophores, calculated as a product of the extinction coefficient and quantum yield, is annotated as numbers adjacent to the respective emission spectra. The spectra and brightness values were adapted from FPbase (Lambert, 2019). (C) Practical visibility of fluorophores observed under a fluorescent stereomicroscope with different filter sets in live worms on NGM plates. Top, widefield images of worms tagged with the indicated fluorophore (FP) imaged through the indicated filter contrasted to non-tagged worms and background. Bottom, schematic illustration of observed view by eye under fluorescent stereomicroscope during screening. mRNA expression of the tagged protein in young adult worms is indicated in FPKM.
Figure 2
Selection of genes and predicted transcript expressions across tissues.
(A) Location of selected genes on Caenorhabditis elegans chromosomes. Numbers in parentheses indicate the injection pool the gene was assigned to. Color of text indicates the fluorophore assigned to the gene: blue, mTagBFP2; green, mStayGold; red, mScarlet3. (B) Transcript expression levels of the selected genes across major tissues of C. elegans as measured by single-cell RNA sequencing of adult hermaphrodites. Data was adapted from the CeNGEN adult hermaphrodite set (Taylor et al., 2021).
Figure 3
Successful tagging across loci, fluorophores, and gene pools.
(A) Total number of targets successfully tagged. White bar denotes all three genes within the pool were successfully tagged in any combination. In four pools, all three targets were isolated in a single, triple-tagged individual worm. In one pool, we obtained all three targets but in doubles. (B) Total insertion efficiencies per locus for the indicated targets across injection pools. Dots, fraction of F1s positive for indicated fluorophore per injected P0. Five worms were injected per pool except for pools 4, 6, and 10, which were repeated after initial failures as shown in panel C. Data shown in panel B only represents the repeat runs and omits the original failed injections. For group 5 including cox-6B::mTagBFP2, worms with detectable levels of mTagBFP2 fluorescence were not recovered in the F1 generation but were isolated among progeny of F1s positive for mStayGold and mScarlet3; we were thus unable to quantify efficiency for this locus at F1. Bars, mean ± SD. (C) Viable brood size of injected P0 worms represented as individual points. Lines, mean ± SD. Numbers above plots, number of successfully isolated tags out of three within the pool. 4 R, 6 R, and 10 R are re-injections of groups 4, 6, and 10. (D) Correlation of viable brood of P0s with fraction of F1 worms positive for any fluorophore tags. Goodness of fit is represented as an R2 value from a simple linear regression. Slope was not significantly greater than zero (p=0.2497, F-test on Prism).
Figure 4
Expression and localization of genes in pool 2.
Expression and localization patterns of translation elongation factor 1 alpha 1 EEF-1A.1::mTagBFP2, nuclear protein 1 homolog ZK632.9::mStayGold, and acyl-CoA dehydrogenase ACDH-10::mScarlet3 proteins in a live adult hermaphrodite worm by confocal microscopy. All three tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Germline and intestine of the animal are delineated in magenta and white dashed lines, respectively. The worm was imaged in separate overlapping frames with high magnification then stitched to reconstruct the whole animal. A single plane near the mid-plane of the animal was imaged.
Figure 5
Expression and localization of genes in pool 3.
Expression and localization patterns of 14-3-3 protein PAR-5::mTagBFP2, heat shock protein family E Y22D7AL.10::mStayGold, and the glucokinase HXK-1::mScarlet3 proteins in a live adult hermaphrodite worm by confocal microscopy. All three tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal.
Figure 6
Expression and localization of genes in pool 9.
(A) Expression and localization patterns of glutamate dehydrogenase GDH-1::mTagBFP2, lysyl-tRNA synthetase KARS-1::mStayGold, and the CREB binding protein CBP-1::mScarlet3 proteins in a live adult hermaphrodite worm by confocal microscopy. All three tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal. (B) Germline insets showing non-overlapping localizations of GDH-1 and KARS-1, predicted mitochondrial proteins. (C) Left, maximum entropy thresholds of GDH-1 and KARS-1 signals. Right, correlation plot of signal intensities of GDH-1 and KARS-1.
Figure 7
Expression and localization of genes in pool 10.
(A) Expression and localization patterns of nucleosome assembly protein NAP-1::mTagBFP2, phosphoribosyl transferase HPRT-1::mStayGold, and ATP citrate lyase ACLY-1::mScarlet3 proteins in a live adult hermaphrodite worm by confocal microscopy. All three tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal. (B) Expressions and localizations of nucleosome assembly protein NAP-1::mTagBFP2, phosphoribosyl transferase HPRT-1::mStayGold, and ATP citrate lyase ACLY-1::mScarlet3 proteins in the head of a live adult hermaphrodite worm by confocal microscopy. Image shows a maximum intensity projection of a Z-stack optically sectioned at 1 µm per stack.
Figure 8
Expression and localization of genes in pool 5.
(A) Expression and localization patterns of cytochrome C oxidase subunit 6B COX-6B::mTagBFP2 and TDO-2::mStayGold proteins in a live adult hermaphrodite worm by confocal microscopy. The two tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal. (B) Expressions and localizations of tryptophan 2,3-dioxygenase TDO-2::mStayGold and hydroxyacyl-CoA dehydrogenase F54C8.1::mScarlet3 proteins in a live adult hermaphrodite worm by confocal microscopy. The two tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal. (C) F54C8.1::mScarlet3 signal in sperm compared to untagged N2 worms imaged under the same settings. (D) F54C8.1::mScarlet3 signal in gut granules compared to untagged N2 worms imaged under the same settings. N=20 F54C8.1::mScarlet3 granules and 24 N2 granules quantified from 2 worms for each genotype. Lines, mean ± SD.
Figure 9
Expression and localization of genes in pool 7.
Expression and localization patterns of heat shock protein family A HSP-1::mTagBFP2 and triosephosphate isomerase TPI-1::mStayGold proteins in a live adult hermaphrodite worm by confocal microscopy. The two tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal.
Figure 10
Expression and localization of genes in pool 8.
(A) Expression and localization patterns of ATPase H+transporting V0 subunit C VHA-2::mTagBFP2 and histone acetyltransferase HAT-1::mScarlet3 proteins in a live adult hermaphrodite worm by confocal microscopy. The two tags were isolated within the same individual then homozygosed at the F2 generation before imaging. Magenta and white dashed lines, germline and intestine, respectively. Image reconstructed from multiple overlapping high-magnification images. Single plane near the mid-plane of the animal. (B) Inset showing germ cell nuclei. The proton pump VHA-2 localizes to nuclear membranes in germ cells specifically, indicated by white arrows.
Tables
Table 1
Summary of tagging approaches and results.
Expected expression patterns are based on indicated single-cell RNA sequencing datasets and are compared to observed patterns of the tagged proteins. Only strains with multiple tags were imaged, to test feasibility of co-imaging across expression and brightness levels without signal bleed-through. Single tagged strains (*) and pool 6 which was heterozygous at time of experiments (**) were not imaged.
| Pool | Locus | Ontology | Tagging | Bulk RNA (FPKM) | scRNA-seq tissues with highest transcript levels | Observed expression | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| modENCODE | CeNGEN/Taylor et al. young adult expression (scaled TPM/Z-score) | Gao et al. young adult expression (Seurat ‘average expression’) | Ghaddar et al. young adult expression (log2(TPM)) | ||||||||||||||||
| Gene | Chr | Class | Function | Term. | FP | Success? | RNAi phen. | SM visible? | L1 | L4 | YA | Tissue | Exp. | Tissue | Exp. | Tissue | Exp. | ||
| 1 | Y82E9BR.3 | III | ATP synthase membrane subunit c | ATP synthesis & ETC | N | mTBFP2 | No | Let | 5484 | 7295 | 3002 | Pharynx (marginal cell) Muscle (body wall) Epidermis (head) | 7.7 3.2 3.1 | Spermatheca Coelomocyte Germline | 16.8 10.9 10.8 | Muscle (pharynx) Somatic gonad (sheath) Muscle (body wall) | 13.4 12.9 12.9 | Not obtained | |
| cts-1 | III | Citrate synthase | Citrate cycle TCA cycle | C | mSG | Yes | Let | Yes | 937 | 466 | 590 | Pharynx (marginal cell) Glia (PHso) Epidermis (seam) | 4.7 3.6 2.7 | Spermatheca Pharynx Intestine | 2.8 2.6 2.4 | Pharynx (marginal) Muscle (pharynx) Intestine | 10.9 10.6 10.3 | Not imaged* | |
| cah-4 | X | Carbonic anhydrase | Nitrogen metabolism | C | mSc3 | Yes | Let | Yes | 209 | 105 | 140 | Epidermis Excretory cell Pharynx (epithelial) | 6.5 6.0 5.6 | Epidermis Gonadal sheath Neuron | 2.7 0.73 0.5 | Excretory cells Neurons (RIB) Epithelium (pharynx) | 10.2 9.4 9.2 | Not imaged* | |
| 2 | eef-1A.1 | III | Elongation factor 1 alpha 1 | Translation | C | mTBFP2 | Yes | Let | Yes | 3491 | 6059 | 7745 | Epidermis (seam) Germline Excretory gland | 5.3 5.2 4.8 | Coelomocyte Spermatheca Intestine | 37.2 33.3 24.7 | Germline Somatic gonad (sheath) Intestine | 14.2 13.1 13.0 | Ubiquitous; high germline enrichment |
| ZK632.9 | III | Nuclear protein 1 | Nucleus | N | mSG | Yes | Stp | Yes | 537 | 561 | 629 | Somatic gonad Pharynx (marginal cell) Epidermis | 6.6 4.1 2.2 | Pharynx Germline Hypodermis | 2.3 2.1 1.9 | Spermatheca Pharynx (marginal cell) Muscle (pharynx) | 11.0 10.3 10.2 | Ubiquitous, even | |
| acdh-10 | X | Acyl-CoA dehydrogenase | Metabolism | C | mSc3 | Yes | Ste | Yes | 135 | 74 | 38 | Epidermis (tail) Muscle (body wall) Rectal cell | 5.6 3.9 3.0 | Muscle Vulva/uterus Pharynx | 0.6 0.5 0.5 | Intestine Muscle (body wall) Spermatocyte | 6.5 6.3 6.2 | Somatically even; germline low | |
| 3 | par-5 | IV | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein | Signaling | C | mTBFP2 | Yes | Let | Yes | 1593 | 3293 | 1568 | Somatic gonad XXX cell Neurons (RIM highest) | 5.3 3.1 2.4 | Germline Sperm Spermatheca | 4.4 4.4 3.3 | Neurons (AWC highest) Germline Intestine | 10.9 10.5 10.4 | Germline highly enriched; neurons observed; elsewhere low |
| Y22D7AL.10 | III | Heat shock protein family E | Proteostasis | C | mSG | Yes | Let | Yes | 426 | 364 | 375 | Germline Glia (PHso) Pharynx (arcade cell) | 7.8 3.9 3.4 | Germline Sperm Glia | 1.8 1.8 1.7 | Germline Intestine Excretory cells | 10.0 9.6 9.3 | Germline enriched; somatically broad | |
| hxk-1 | I | Glucokinase | Glycolysis / Gluconeogenesis | C | mSc3 | Yes | Let | Yes | 54 | 46 | 117 | Neurons (DB highest) Pharynx (marginal cell) Coelomocyte | 4.0 2.4 2.3 | Somatic gonad (sheath) Coelomocyte Neurons | 5.0 3.4 2.8 | Coelomocyte Various neurons (DB or VB highest) | 9.8 9.8 | High gonadal sheath enrichment; elsewhere low | |
| 4 | act-4 | X | Actin beta | Cytoskeleton | C | mTBFP2 | No | Let | 1895 | 1662 | 883 | Somatic gonad Muscle (body wall) Neurons (PVD highest) | 12 2.9 2.7 | Spermatheca Muscle Pharynx | 14.7 13.2 7.7 | Muscle (vulval highest) Neurons (PVD or FLP) Spermatheca | 13.9 13.1 12.8 | Not obtained | |
| pyr-1 | II | Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase | Pyrimidine metabolism | C | mSG | Yes | Let | Yes | 17 | 59 | 15 | Somatic gonad (sheath) Neurons (URY) Excretory gland cell | 5.9 5.4 1.9 | Somatic gonad (sheath) Coelomocyte Elsewhere low | 4.3 2.5 | Somatic gonad (sheath) Neurons (LUA highest) Intestinal valve | 9.7 7.5 7.2 | Not imaged* | |
| fasn-1 | I | Fatty acid synthase | Fatty acid biosynthesis | C | mSc3 | No | Let | 32 | 38 | 43 | Epidermis (seam) Glia (socket) Somatic gonad (sheath) | 7.9 5.5 4.8 | Somatic gonad (sheath) Epidermis Glia | 21.1 2.1 1.2 | Somatic gonad (sheath) Epidermis (seam) Glia | 11.2 9.3 8.5 | Not obtained | ||
| 5 | cox-6B | III | Cytochrome c oxidase subunit 6B | ATP synthesis & ETC | C | mTBFP2 | Yes | Let | Dim F1, F2 yes | 932 | 1650 | 857 | Pharynx (marginal cell) Somatic gonad (sheath) Intestine | 3.9 3.4 3.2 | Sperm Spermatheca Glia | 5.2 5.0 4.8 | Muscle (pharyngeal) Pharynx (marginal cells) Somatic gonad (sheath) | 11.2 10.7 10.6 | Ubiquitous; distal germline, intestine enriched |
| tdo-2 | III | Tryptophan 2,3-dioxygenase | W degradation | C | mSG | Yes | Gro | Yes | 132 | 201 | 89 | Epidermis Excretory (gland cell) Pharynx (gland) | 10 3.3 1.0 | Epidermis Elsewhere low | 3.1 | Excretory duct Pharynx (gland) Epidermis | 8.6 7.3 7.1 | Epidermis enriched or specific | |
| F54C8.1 | III | Hydroxyacyl-CoA dehydrogenase | Fatty acid elongation | C | mSc3 | Yes | Let | Yes | 1 | 34 | 16 | Sperm Elsewhere low | 13 | Sperm Elsewhere low | 0.6 | Sperm Elsewhere low | 8.3 | Sperm enriched or specific | |
| 6 | pab-1 | I | Poly(A) binding protein | Transcript regulation | C | mTBFP2 | Yes | Let | Yes | 2000 | 1017 | 932 | Neurons (PDB highest) Pharynx Epidermis (seam) | 5.3 4.0 2.7 | Coelomocyte Sperm Germline | 16.5 10.9 9.1 | Germline Uterus Rectal gland | 11.8 10.6 10.5 | Not imaged** |
| dld-1 | IV | Dihydrolipoamide dehydrogenase | Glycolysis / Gluconeogenesis | C | mSG | Yes | Let | Yes | 191 | 200 | 202 | Epidermis (head) Muscle (pharynx) Muscle (body wall) | 3.9 3.4 3.1 | Germline Sperm Coelomocyte | 1.9 1.7 1.6 | Neurons (M4) Pharynx (epithelial) Muscle (pharynx) | 8.7 8.6 8.1 | Not imaged** | |
| set-1 | III | Lysine methyltransferase | Histone modification | C | mSc3 | No | Let | 33 | 27 | 58 | Neurons (AWB highest) Germline Epidermis | 9.7 3.9 1.9 | DTC or excretory gland Germline Sperm | 1.2 1.1 1.0 | Neurons (AWB highest) Oocytes Sperm | 6.9 6.8 6.2 | Not obtained | ||
| 7 | hsp-1 | IV | Heat shock protein family A | Proteostasis | C | mTBFP2 | Yes | Let | Yes | 376 | 999 | 1377 | Neurons (PQR highest) Somatic gonad (DTC) XXX cell | 3.7 3.5 2.1 | Sperm Germline Coelomocyte | 8.5 7.3 6.3 | Germline Neurons (ASI highest) Excretory cell | 10.5 10.2 9.4 | Ubiquitous; germline enriched |
| tpi-1 | II | Triosephosphate isomerase | Glycolysis / Gluconeogenesis | C | mSG | Yes | Yes | 262 | 180 | 248 | Excretory cell Neurons (RIA highest) Epidermis | 5.1 2.7 2.2 | Vulva/uterus Glia Sperm | 0.7 0.6 0.6 | Neurons (RMD highest) Muscle (body wall) Pharynx (epithelium) | 10.2 8.1 8.0 | Ubiquitous; neuron enriched | ||
| flad-1 | II | Flavin adenine dinucleotide synthetase | Riboflavin metabolism | Exon 4 | mSc3 | No | Gro | 18 | 24 | 30 | Neurons (PHA highest) Germline Intestine | 6.3 5.7 3.4 | Sperm Germline Neurons | 1.2 1.1 0.6 | Germline Somatic gonad (male) Intestine | 6.0 5.9 5.5 | Not obtained | ||
| 8 | vha-2 | III | ATPase H+transporting V0 subunit c | Proton transport | C | mTBFP2 | Yes | Let | Yes | 1565 | 873 | 799 | Excretory cell Neurons (VC4/5 highest) Glia (AMso) | 8.7 6 3.3 | Neurons Glia Uterine seam | 2.9 2.8 2.8 | Neurons (VC4/5 highest) Glia (AMso) Excretory cells | 12.3 11.6 11.4 | Ubiquitous, even |
| asb-1 | III | ATP synthase peripheral stalk-membrane subunit b | Oxidative phosphorylation | N | mSG | No | Let | 25 | 157 | 266 | Germline Excretory cell Neurons (PQR highest) | 11 3.7 3.0 | Sperm Germline Spermatheca | 1.8 1.4 0.9 | Sperm Germline Oocytes | 9.4 9.3 8.5 | Not obtained | ||
| hat-1 | III | Histone acetyltransferase 1 | Histone modification | C | mSc3 | Yes | Let | Dim but visible at F1 | 22 | 23 | 56 | Germline Epidermis Coelomocyte | 8.0 3.5 2.8 | Germline Sperm Somatic gonad (DTC) | 0.8 0.8 0.6 | Germline Pharynx (gland) Neurons (AFD highest) | 5.6 5.5 5.2 | Ubiquitous; germline enriched | |
| 9 | gdh-1 | IV | Glutamate dehydrogenase | Glutamate / glutamine metabolism | C | mTBFP2 | Yes | Ste | Yes | 921 | 565 | 869 | Pharynx (epithelium) Muscle (hmc) Glia (AMso) | 5.0 3.7 3.0 | Spermatheca DTC or excretory gland Intestine | 5.5 5.5 5.1 | Muscle (pharynx) Pharynx (marginal cells) Intestine | 10.6 10.4 10.0 | Ubiquitous, even |
| kars-1 | II | Lysyl-tRNA synthetase | Aminoacyl-tRNA biosynthesis | C | mSG | Yes | Let | Yes | 174 | 153 | 246 | Germline Excretory cell Rectal gland | 10 2.7 2.4 | Sperm Germline Neuron | 1.6 1.5 1.2 | Germline Sperm Intestine | 9.0 8.1 7.9 | Germline enriched; elsewhere low | |
| cbp-1 | III | CREB binding protein | Histone modification | C | mSc3 | Yes | Let | Dim but visible at F1 | 41 | 23 | 27 | Neurons (VA1 highest) Glia (socket) Somatic gonad (DTC) | 2.3 1.2 0.8 | Coelomocyte Pharynx Uterine seam | 4.0 3.5 3.4 | Intestine Neurons (NSM) Somatic gonad (sheath) | 9.1 9.0 8.4 | Somatically even; germline depleted | |
| 10 | nap-1 | IV | Nucleosome assembly protein | Nucleus | C | mTBFP2 | Yes | Let | Yes | 630 | 501 | 546 | Somatic gonad (DTC) Pharynx (epithelium) Epidermis (seam) | 7.2 6.3 3.6 | Sperm Germline Coelomocyte | 4.3 3.3 2.7 | Germline Excretory cells Somatic gonad (sheath) | 10.8 9.6 9.5 | Ubiquitous; germline enriched |
| hprt-1 | I | Phosphoribosyl transferase | Purine metabolism | C | mSG | Yes | Yes | 61 | 101 | 118 | Glia (sheath) Glia (CEPsh) Intestine | 9.9 8.4 2.3 | Intestine Somatic gonad (sheath) Vulva/uterus | 1.6 1.0 0.8 | Glia (CEPsh) Intestine Gonadal sheath | 10.9 10.1 9.5 | Glia, intestine enriched; elsewhere low or absent | ||
| acly-1 | X | ATP citrate lyase | Citrate cycle TCA cycle | N | mSc3 | Yes | Let | Yes | 11 | 18 | 43 | Neurons (RMD highest) Neurons (VB2) Elsewhere low | 4.4 4.1 | DTC or excretory gland Coelomocyte Elsewhere low | 1.3 0.5 | Neurons (RMD highest) Neurons (PHC) Elsewhere low | 9.4 8.8 | Neuron enriched; elsewhere low | |
Additional files
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Supplementary file 1
Catalog of primers, CRISPR RNAs and fluorophore sequences used.
Tab 1, sequences of amplification primers used to introduce locus-specific homology arms and generate repair templates; tab 2, sequences of CRISPR RNAs (crRNAs) used to edit indicated loci; tab 3, sequences of the fluorescent protein-encoding DNA fragments used as amplification templates to generate repair templates.
- https://cdn.elifesciences.org/articles/110717/elife-110717-supp1-v1.xlsx
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Supplementary file 2
Source data for plots.
Source numerical data for plots in Figures 3B, C, D, 8D.
- https://cdn.elifesciences.org/articles/110717/elife-110717-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110717/elife-110717-mdarchecklist1-v1.docx
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A pilot study for whole proteome tagging in Caenorhabditis elegans
eLife 15:RP110717.
https://doi.org/10.7554/eLife.110717.4
