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) Schematic of the practical visibility of fluorophores observed under a fluorescent stereomicroscope with different filter sets in live worms on NGM plates.

Selection of genes and predicted transcript expressions across tissues.

(A) Location of selected genes on C. 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).

Genes tagged

Successful tagging across loci, fluorophores and gene pools.

(A) Total number of targets successfully tagged. White bar denotes all 3 genes within the pool were successfully tagged in any combination. In four pools, all 3 targets were isolated in a single, triple-tagged individual worm. In one pool, we obtained all 3 targets but in doubles. (B) Total insertion efficiencies 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, 10 and 6 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. 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 3 within the pool. 4R, 6R and 10R 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).

Expression and localization of genes in pool 2.

Expressions and localizations 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.

Expression and localization of genes in pool 3.

Expressions and localizations 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.

Expression and localization of genes in pool 9.

(A) Expressions and localizations 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.

Expression and localization of genes in pool 10.

(A) Expressions and localizations 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. 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.

Expression and localization of genes in pool 5.

(A) Expressions and localizations 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.

Expression and localization of genes in pool 7.

Expressions and localizations 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.

Expression and localization of genes in pool 8.

(A) Expressions and localizations 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.