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A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion

Abstract

Apicomplexan parasites, such as Toxoplasma gondii, have specific adaptations that enable invasion and exit from the host cell. Owing to the phylogenetic distance between apicomplexan parasites and model organisms, comparative genomics has limited capacity to infer gene functions. Further, although CRISPR/Cas9-based screens have assigned roles to some Toxoplasma genes, the functions of encoded proteins have proven difficult to assign. To overcome this problem, we devised a conditional Cas9-system in T. gondii that enables phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we screened 320 genes to identify those required for defined steps in the asexual life cycle. The detailed characterization of two genes identified in our screen, through the generation of conditional knockout parasites using the DiCre-system, revealed that signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, and a novel conoid protein (conoid gliding protein, CGP) functions late during egress and is required for the activation of gliding motility. Establishing different indicator lines and applying our conditional Cas9 screen could enable the identification of genes involved in organellar biogenesis, parasite replication or maintenance of the endosymbiotic organelles in the future.

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Fig. 1: Adaptation and characterization of the splitCas9-system in T. gondii.
Fig. 2: Phenotypic screen for actin dynamics, apicoplast segregation and egress mutants.
Fig. 3: Analysis of cgp and slf.
Fig. 4: SLF and CGP act at different times during egress.
Fig. 5: SLF is a crucial part of the signalling platform.
Fig. 6: CGP localizes to the preconoidal rings and egress models.

Data availability

All imaging data generated and used for this paper are available from the authors on reasonable request. Expression constructs and parasite strains have been deposited in Addgene (https://www.addgene.org/). Information on T. gondii genes and proteins were obtained in ToxoDB (release 30 to 56). Data obtained from sequencing have been deposited in SRA (https://www.ncbi.nlm.nih.gov/sra) Bioproject ID: PRJNA821386.Source data are provided with this paper.

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Acknowledgements

We thank all colleagues who contributed antibodies and reagents for this study. In particular, we thank the Lourido lab (Whitehead Institute for Biomedical Research) for assisting with the design of the sgRNA library and many useful discussions. W.L. was funded by a CSC fellowship (201806910075). This project was funded within the DFG Priority Programme SPP2225, Project ME 2675/7-1.

Author information

Authors and Affiliations

Authors

Contributions

W.L. identified egress mutants and performed phenotypic assays for SLF and CGP. J.G. designed and performed the phenotypic screen, identified and tagged candidate genes and assisted with phenotyping. J.F.S. established and characterized the sCas9-system in T. gondii, designed the sgRNA library and analysed egress for ADF. M.G. assisted with phenotyping and analysis of parasites. J.P. assisted in experiment design and data analysis. M.M. designed and coordinated the project and experiments, analysed the data, contributed resources and wrote the paper. E.J-R. designed and coordinated the project and experiments, analysed the data and wrote the paper.

Corresponding authors

Correspondence to Markus Meissner or Elena Jimenez-Ruiz.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Microbiology thanks Mohamed-Ali Hakimi, Christopher Tonkin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Generation of parasite strains RHsCas9, RH-gap40, RHsCas9-gap40, RHsCas9-sag1, RHsCas9-drpA, RHsCas9-act-1 and RHsCas9-adf.

a, Scheme of expression cassettes for the N- and C-termini of the Cas9 enzyme (split 4 variant, see2). Arrows indicate PCR amplicon for verification of plasmid integration (see (b)). b, Analytical PCR confirming integration of sCas9 plasmids into the genome of indicated parasites. c, Scheme of the expression cassette for the single-guide RNA (sgRNA, here for targeting of gap40). Arrows indicate PCR amplicon for verification of plasmid integration. d, Analytical PCR confirming integration of gap40-sgRNA-plasmid into the parasite genome. e, Analytical PCRs confirming integration of sCas9 plasmids into the RH parasites. f, Analytical PCRs confirming integration of indicated sgRNA-plasmids into the parasite genome. g, Validation of specific introduction of indels at the sgRNA cut side in indicated parasites. Cultures were induced with 50 nM rapamycin for 1 h. Parasites were grown for 48 h prior to gDNA collection. The sgRNA cut site was amplified by PCR and sequenced. Red letters represent nucleotide insertion in the mutant strain, causing a frame shift and, thus, the functional disruption of the indicated gene. Black arrows indicate the predicted cut site.

Source data

Extended Data Fig. 2 The nuclear phenotype is caused by Cas9-mediated double strand breaks.

a, Disruption of sag1 in RH and RHΔku80 parasites using a transient assay. RH and RHΔku80 parasites were transiently transfected with a Cas9-YFP-sag1sgRNA plasmid. Parasites were fixed 48 h after transfection. Only parasites that had been transfected successfully (as determined by expression of Cas9-YFP) were counted. Transfection efficiencies were 21.5% (±4.8) for RHΔku80 and 13.2% (±3.6) for RH parasites. A total of n = 129 (RHΔku80) and n = 79 (RH) parasites were counted over 3 biologically independent experiments. b, Genome sequencing verified disruption of sag1 in a clonal RHsCas9-Δhx-sag1-KO mutant. Green letters indicate sgRNA sequence. Red letters represent nucleotide insertions upon disruption of sag1, causing frame shifts. The black arrow indicates the predicted cut side. c, Schematic of the strains sag1KO-sag1* and sag1wt-sag1*. Both strains were generated in the RHsCas9-sag1 background. The endogenous sag1 gene in the sag1KO-sag1* line has been disrupted by prior sCas9 activation. The endogenous sag1 gene of sag1wt-sag1* is intact. The additional sag1 copy has been modified as indicated (red letters) to be resistant to sag1sgRNA recognition. d, IFA depicting the impact of sCas9 activation on sag1KO-sag1* and sag1wt-sag1* parasites. Parasites were induced ± 50 nM rapamycin for 1 h. Scale bars are 5 µm. e, f, quantification of vacuoles displaying aberrant nuclei and cellular morphology after sCas9 activation at 48 h post inoculation. e, quantification of aberrant vacuoles when targeting sag1 in sag1KO-sag1* and sag1wt-sag1* strains. For each condition at least 300 vacuoles were counted in 3 biologically independent assays. Data presented as Mean ± SD. Dots represent the Mean of each replicate, f, quantification of aberrant vacuoles in all controls employed to validate the strain used in the screen. A minimum of 300 vacuoles per condition and per strain were counted over 3 biologically independent assays. Statistics: one-sided ANOVA with Tukey’s multiple test for comparison was used to analyse quantification data. Data presented as Mean ± SD.

Source data

Extended Data Fig. 3 Characterisation of egress in parasites with depleted ADF.

a, Images depict intracellular and egressed vacuoles with or without intravacuolar filamentous actin structures. For this experiment, indicated parasites were induced (50 nM rapamycin, 1 h) or non-induced and grown for 48 h. Egress was then induced by incubating parasites with 2 µM calcium ionophore (Ci) A23187 for 8 min. Scale bars are 5 µm. b, Rapamycin induced (50 nM, 1 h, upper panel) and non-induced parasites (lower panel) were grown for 48 h. Egress was then triggered by incubating parasites with 2 µM Ci A23187 for 8 min. Rate of egress of indicated induced parasites (upper panel) was normalized to egress of the corresponding non-induced parasite population (lower panel). For each condition at least 300 vacuoles were counted over 3 biologically independent replicates. In one of the three biological repeats for RHsCas9-adf, parasites were induced for 48 h and egress was initiated with Ci A23187 for 5 min. Data were presented as Mean ± SD. Dots represent the mean of each replicate. c, Analysis of the abundance of large intravacuolar filamentous actin structures after induction of egress in sag1-wt/KO and adf-wt/KO parasites. See (a) for representative images. Analysis is based on 3 biologically independent replicates. Statistics: one-sided ANOVA with Tukey’s multiple test for comparison was used to analyse quantification data. Data presented as Mean ± SD.

Source data

Extended Data Fig. 4 Selection of candidates and super-aberrant phenotype of parasites with multiple sgRNAs.

a, Parasites containing the sgRNA targeting prf (positive control for F-actin dynamic phenotype) were isolated multiple times and presented a thick, extensive intravacuolar network. Scale bar is 3 μm. This phenotype is very similar to the one observed after the depletion of ADF. b, Parasites containing the sgRNA targeting gap40 (positive control for replication phenotype). The identified phenotype is identical to the one reported previously7. Scale bar is 5 μm. c-g Example of images obtained in the screen. Parasites after 48 hours of induction with 50 nM rapamycin. c, Parasites presenting no phenotype. Note that vacuoles with aberrant shape or nuclei can be found but in low percentage. d, Parasites presenting aberrant morphology. Authors classified this specific well as “gap40KO-like vacuoles”. gap40 gRNA was confirmed after sequencing. e, Clone classified as phenotype affecting nuclei. This clone, and similar clones, were discarded for further analysis. f, Parasites presenting impaired replication. This clone, and similar clones, were discarded for further analysis. g, Parasites presenting an apicoplast related phenotype. h, Parasites with altered F-actin dynamics in the intra vacuolar network. Zoom-in squares shows nuclei in detail (DAPI stain) White squares: vacuoles with aberrant morphology and/or nuclei; Purple squares: vacuoles with specific phenotype. Images taken with a 20x objective. Scale bars are 10 μm. i, Some of the most impressive phenotypes discovered in the screen were caused by multiple integration of sgRNA targeting different genes. Super-resolution image of a representative clone is shown. Scale bar is 3 μm. These clones were omitted from further analysis. This figure shows representative images of at least 2 replicates with same results with the exception of screen derived images where 3 images were obtain from the same well. Selected candidates were then induced and imaged again to verify the observation in the screen.

Extended Data Fig. 5 Mutants with (slight) changes in F-actin localisation and apicoplast maintenance.

a, Mutants with F-actin phenotype. b, Mutants with apicoplast phenotypes. c, Mutants with both F-actin and apicoplast phenotypes. Left images: Images of sCas9-CbEm-FNR clones containing sgRNAs targeting indicated genes. Parasites were induced for 48 h with rapamycin before fixing and imaging. CbEm, STED images. FNR and Hoechst are confocal images. Right images: Widefield images of RHΔku80 parasites with the respective candidate gene endogenously tagged as indicated. Scale bars are 5 μm. This figure shows representative images of at least 2 biologically independent replicates with same results.

Extended Data Fig. 6 Validation of candidates.

a, Quantification of induced egress in RHsCas9-CbEm-FNR-RFP isolated clones with 2 µM of calcium ionophore (Ci) A23187. Parasites were pretreated ±rapamycin for 48 hours prior to induction of egress. Results of the rapamycin (R) induced parasite (+R + Ci) were standardized to the non-rapamycin treated condition (-R + Ci). Red dash line marks reduction of egress to 50% when compared to non-induced parasites. 4 candidates were selected after this analysis. A minimum of 300 vacuoles per condition and per strain were counted over 3 biologically independent replicates. Statistic data show the comparison to the RHsCas9 strain. Data presented as relative Mean + SD. Dots represent the mean of each replicate. b, Representative images of the 4 candidate genes and control parasites, as quantified in (a). Scale bar, 30 μm. c, 24-hour replication assay. Vacuoles were counted and number of parasites per vacuole was determined. Quantification of parasite replication showed a significant delay in replication for TGGT1_252465, which was omitted from further analysis. A minimum of 280 vacuoles per condition and per strain were counted over 3 biologically independent assays. Data presented as Mean + SD. Colour-coded P values represent the condition analysed. d, Quantification of invasion of indicated parasites was standardized to the non-rapamycin treated condition and normalized to RHsCas9 strain. Parasites were pretreated ± 50 nM rapamycin for 48 hours before the invasion assays were carried out. Statistics are compared to RHsCas9 strain. Vacuoles per condition and per strain counted over 3 biologically independent replicates. Data were presented as Mean + SD. Dots represent the mean of each replicate. Unpaired two-tailed Student’s t-test was calculated for all quantifications.

Source data

Extended Data Fig. 7 Generation of parasite lines in DiCre background parasites.

a, C-terminal tagging scheme. Donor DNA containing respective tag, loxP sequence and 50 nucleotides of homologous flanking sequence was co-transfected with gRNA-Cas9YFP vector. Primers 1 and 1’ bind outside of the homologous region incorporated in donor DNA and were used to verify insertion of the tag. Orange arrow indicates the targeted region of the sgRNA. b, Generation of floxed lines. Insertion of upstream loxP sequence before the open reading frame at 5’UTR region was achieved by co-transfection of gRNA-Cas9YFP together with a long oligo containing a loxP sequence flanked by 33 nt of homology to the targeted region. Primers 2 and 3’ were used for verification of loxP integration. Primers 3 and 3’ bind outside of the homologous region flanking the loxP and were used for sequencing of the insertion. Primers 3 and 1’ were used for confirmation of gene excision. Orange arrow indicates the targeted region of the sgRNA. c, Analytical PCR shows the correct integration of tags in the indicated parasite lines. d, Integration PCR indicates the loxP insertion at the 5’UTR in the indicated parasite lines. e, Genotyping PCR verifying the loxP insertion at 5’UTR in the indicated parasite lines. These amplicons were sent for sequencing to confirm correct sequences. f, PCR indicating excision of the floxed gene. g, Correct integration of 6HA and eGFP for RNG2 and SAS6L in loxPcgp-Halo parasite lines, respectively. h, PCR shows the correct integration of tags in the floxed parasite lines. i, Genotyping PCR indicates successful tagging of gc, cdc50.1, and ugo. j, Genotyping PCR indicates the loxP insertion at 5’UTR in the indicated parasite lines. k, Scheme for replacing UPRT locus with CbEm driven by Tgdhfr promotor. Orange arrow indicates the targeted region of the sgRNA. Primers 4 and 4’ were used to verify insertion of the tag. Primers 4 and 5 were also used to verify insertion of the tag. l, PCRs showing integration and for genotyping of CbEm in floxed parasite lines.

Source data

Extended Data Fig. 8 Deletion of SLF does not interfere with microneme or rhoptry trafficking.

a, SLF localized at the apical tip and the intravacuolar network, as shown by co-localisation with CbEm. Scale bar, 5 µm. b, Quantification of the percentage of vacuoles without SLF signal in loxPslf-mCherry parasites treated with 50 nM rapamycin. At least 300 vacuoles were counted over 3 biologically independent replicates. c, Parasites lacking slf showed a normal intracellular replication. For each condition, at least 300 vacuoles were counted over 3 biologically independent replicates. Dots represent the mean of each replicate. d, Invasion-attachment assay in the presence of 2 µM calcium ionophore A23187 after 96 hours post induction. Data was normalized to DiCreΔKu80 parasites. 3 biological replicates were performed. e, The gliding length (i) and average gliding speed (ii) of parasites capable of gliding (helical or circular movement) was measured by tracking 18 parasites in the presence of 2 μM Ci A23187 after 96 hours post induction ± rapamycin. Data presented is Mean + SEM. Dots represent the value for each parasite tracked. f. Trail deposition assay of loxPslf-mcherry parasites pretreated ± 50 nM rapamycin for 96 hours stimulated ±2 μM Ci A23187. g-i, Apicoplast, microneme and rhoptry proteins are not affected upon deletion of slf at 72-hour post induction ± rapamycin. HSP60, marker for apicoplast. MIC2 and AMA1: markers for microneme proteins. ROP1 and ROP2-4: markers for rhoptry proteins. Scale bar: 5 µm. j, Microneme secretion assay performed on wildtype (WT) parasites and loxPslf-mCherry. Parasites were pretreated ± 50 nM rapamycin for 72 hours. Triangles indicate the unprocessed (blue) and processed (red) form of MIC2. This assay was performed in 3 biological replicates. Quantifications are shown in Fig. 3g. Statistics: unpaired two tailed Student’s t-test were calculated. Colour-coded P values represent the condition compared. Data were presented in all graphs as Mean + SD unless otherwise specified.

Source data

Extended Data Fig. 9 Loss of CGP doesn’t affect parasite replication or secretory organelles.

a, Quantification of the percentage of vacuoles with loss of CGP signal. loxPcgp-Halo parasites were treated with 50 nM rapamycin for 24 h, 48 h, 72 h, and 96 h. At least 100 vacuoles were counted in each 3 biologically independent replicates. Dots represent the mean of each replicate. b, Parasites lacking cgp showed a normal intracellular replication. For each condition, at least 100 vacuoles were counted in each 3 biologically independent replicates. Replication assays were performed after 96 hours incubation with or without 50 nM rapamycin. c, The gliding length (i) and average gliding speed (ii) of parasites capable of gliding (helical or circular movement) were measured by tracking 18 parasites after 72 hours post induction with rapamycin. Motility was analysed by manual tracking the parasites using Icy software. Data presented as Mean + SEM. Dots represent each parasite tracked. d-f) Localisation of apicoplast, microneme and rhoptry proteins is not affected upon deletion of cgp. Parasites were pre-treated ± 50 nM rapamycin for 1 hour and imaged 72 hours later. HSP60: marker for apicoplast. MIC2, MIC6, and MIC8: markers for microneme proteins. ROP1 and ROP2-4: markers for rhoptry proteins. Scale bar, 5 μm. g, Microneme secretion assay performed on wildtype (WT) parasites and loxPcgp-Halo after 72 hours incubation with or without 50 nM rapamycin. Triangles indicate the unprocessed (blue) and processed (red) form of MIC2. This assay was performed in 3 biological replicates. Quantifications are shown in Fig. 3h. Statistics: Unpaired two tailed Student’s t-test was calculated. Colour-coded P values represent the condition compared. Data were presented in graphs as Mean + SD unless otherwise specified.

Extended Data Fig. 10 Further characterisation of slf-cKO parasites and preliminary data on other sCas9 screens.

a, Non-egressed slf-cKO parasites induced with Ci A23187 are able to disassemble the filamentous network. Representative images of parasites lacking SLF after induction with 2 µM A23187 for 5 min. Egress assay was performed 58 hours post rapamycin induction. Scale bar is 5 µm. b, Quantification of intact vacuoles with no SLF signal after 5 min induction. At least 100 vacuoles were counted per biologically independent replicate (n = 3). Statistics: Unpaired two tailed Student’s t-tests were calculated. Colour-coded P values represent the condition compared. Data presented as Mean + SD. c and d, GABA and GABA analogues effect on parasites with depleted SLF. Plaque assay of loxPslf-mCherry supplemented with different concentration of GABA (c) and Gabapentin (d) indicating SLF does not play a role in GABA signalling in Toxoplasma. Scale bar: 1.5 mm. H2O vehicle control was added in the same volume that the employed at the GABA concentration indicated. e, Alternative indicator strains for alternative screens. Three alternative indicator strains were created to date. Images depicting the parasites strains: sCas9-IMC1_YFP-MIC8_mCherry (IMC and micronemes), sCas9-CbEm-mCherry_αTubulin (mChTub: F-actin and microtubules) and sCas9-CbEm_HSP60-RFP (F-actin and mitochondria). f, Representative images of our main control for the indicator strain sCas9-CbEm-mChTub were a sgRNA targeting α-tubulin was inserted. Parasites showed a collapse vacuole where no microtubules (MT) can be observed, and nuclei are undivided. g, Quantification of aberrant morphology and nuclei in the sCas9-CbEm-mChTub strain containing either α-tubulin-sgRNA or sag1-sgRNA. While parasites with targeted MT present almost a 100% of affected vacuoles, sag1 shows only morphology changes in around 50% of the population. Quantifications done in biologically independent replicates. Statistics: one-sided ANOVA and Tukey’s tests were employed in this quantification. Data presented as Mean ± SD.

Supplementary information

Supplementary Information

Supplementary Discussion

Reporting Summary

Peer Review File

Supplementary Table

Supplementary Table 1. List of gRNAs. Table 2. List of candidates and their gRNAs. Table 3. Candidates picked. Table 4. Candidate genes selected for characterization. Table 5. Oligonucleotides. Table 6. Antibodies and dyes.

Supplementary Video 1

Time-lapse video microscopy of gliding parasites. a, Non-induced loxPcgp-Halo parasites gliding normally. b,c, Gliding in pre-induced loxPcgp-Halo parasites with 50 nM rapamycin for 72 h. Only parasites not expressing CGP were analysed. b, Non-gliding cgp-cKO. c, Gliding cgp-cKO. Note that movement is slow and the distance covered is minimal. d, Non-induced loxPslf-Halo parasites gliding normally. e,f, Gliding in induced loxPslf-Halo parasites with 50 nM rapamycin for 96 h. Only parasites not expressing SLF were analysed. e, In most cases, slf cKO parasites seem to float over the surface. f, Some slf cKO parasites attach on the FCS-coated surface. However, no movement is observed. Time indicated in minutes:seconds. Scale bars, 5 μm.

Supplementary Video 2

Time-lapse video microscopy of loxPslf gliding parasites in the presence of 2µM Ci A23187. a, Non-induced with rapamycin, loxPslf-Halo parasites glide normally. b, Induced KO parasites (only parasites not expressing SLF were analysed; slf-cKO) can glide normally upon addition of Ci A23187. Slf cKO parasites were induced with rapamycin for 96 h before egress induction. Time indicated in minutes:seconds. Scale bars, 5 μm.

Supplementary Video 3

Egress induction in LoxPslf-Halo expressing CbEm parasites (loxPslf-Halo/CbEm). ac, Non-rapamycin induced parasites. a, Normal egress after addition of BIPPO. b, Parasites egress normally after addition of Ci A23187. c, Egress after addition of propranolol. df, Parasites induced with rapamycin for 72 h before egress induction with different compounds (only parasites not expressing SLF were analysed). d, Parasites are unable to initiate egress after addition of BIPPO. Note that the intravacuolar network and the polymerization centre at the Golgi remain intact. e,f, Addition of Ci A23187 partially rescues the egress phenotype previously observed. Note that parasites are able to disassemble F-actin filaments (e) but in some cases are unable to leave the host cell (f). g, Upon addition of propranolol hydrochloride, parasites remain inside the host cell despite being able to disassemble the filamentous network. Videos were recorded at 0.33 frames per second. Time indicated in minutes:seconds. Scale bar, 5 μm.

Supplementary Video 4

Egress induction in LoxPcgp-Halo expressing CbEm parasites (loxPcgp-Halo/CbEm). ac, Non-rapamycin induced parasites. a, Normal egress after addition of BIPPO. b, Parasites egress normally after addition of Ci A23187. c, Egress after addition of propranolol. df, Parasites induced with rapamycin for 72 h before egress induction with different compounds (only parasites not expressing CGP were analysed). d, Although parasites are able to disassemble the F-actin network and the polymerization centre at the Golgi area disappears, they cannot initiate movement to egress from the host cell after addition of BIPPO. e, Addition of Ci A23187 does not rescue the egress phenotype previously observed. f, Upon addition of propranolol hydrochloride, parasites remain inside the host cell. Videos were recorded at 0.33 frames per second. Time indicated in minutes:seconds. Scale bar, 5 μm.

Supplementary Video 5

Parasitophorous vacuole membrane (PVM) integrity of parasites transiently expressing SAG1ΔGPI-dsRed after induction with BIPPO. a,b, dsRed signal diffuses rapidly after initiation of egress in wild-type parasites (non-rapamycin induced), indicating rupture of the PVM before egress. c, In parasites lacking CGP, PVM lyses but parasites are unable to initiate movement. d, Depleted SLF prevents lysis of PVM and movement initiation in BIPPO-induced parasites. F-actin, yellow. SAG1ΔGPI-dsRed, pink. Time interval between frames is 2 s for non-induced parasites, 5 s for cpg-cKO parasites and 10 s for slf-cKO. Time indicated in minutes:seconds. Scale bar, 5 μm.

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Li, W., Grech, J., Stortz, J.F. et al. A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion. Nat Microbiol 7, 882–895 (2022). https://doi.org/10.1038/s41564-022-01114-y

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