Whole genome sequencing across the tree of life has revealed the surprising observation that nine essential amino acid (EAA) biosynthesis pathways are missing from the metazoan lineage (Payne and Loomis, 2006). Furthermore, these losses appear to have occurred multiple times during eukaryotic evolution, including in some microbial lineages (Figure 1A; Payne and Loomis, 2006; Guedes et al., 2011). Branching from core metabolism, the nine EAA biosynthesis pathways missing from metazoans involve over 40 genes (Figure 1B, Supplementary files 1-3) that are widely found in bacteria, fungi, and plants (Guedes et al., 2011). While the absence of pathways that produce essential metabolites is observed in certain bacteria (Zengler and Zaramela, 2018), which possess short generation times and high genomic flexibility to adapt to rapidly changing environments, the forces driving the loss of multiple EAA biosynthetic pathways in multicellular eukaryotes remain a great mystery. An exception that proves the rule is the partial reacquisition of EAA biosynthetic pathways through horizontal gene transfer in certain rare insect lineages which host genome-reduced intracellular bacteria and feed on simple nutrient sources such as sap or blood (Wilson and Duncan, 2015). Recent efforts in genome-scale synthesis (Isaacs et al., 2011; Mitchell et al., 2017; Fredens et al., 2019) and genome-writing (Boeke et al., 2016) have highlighted our increasing capacity to construct synthetic genomes with novel properties, thus providing a route to not only examine these interesting evolutionary questions but also yield new capacities of bioindustrial utility (Heng et al., 2015; Tan et al., 2021; Kitada et al., 2018).

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We sought to explore the possibility of generating prototrophic mammalian cells capable of complete biosynthesis of EAAs using a synthetic genomics approach (Figure 1C). The Chinese hamster ovary (CHO) K1 cell line was chosen as a model system due to its fast generation time, amenability to genetic manipulations, availability of a whole genome sequence, and established industrial relevance for producing biologics (Fischer et al., 2015). EAA biosynthesis genes from the best characterized model organisms were considered during pathway design while optimizing for the fewest number of enzymes needed for a given EAA pathway. To avoid using multiple promoters, we introduced ribosome-skipping 2A sequences Szymczak-Workman et al., 2012 between biosynthetic genes to allow for protein translation of separate enzymes from a single transcriptional unit. The EAA pathway and an additional EGFP reporter were placed in a vector that could be integrated as a single copy into the CHO genome at a designated landing pad using the Flp-In system (O’Gorman et al., 1991). The entire pathway was synthesized de novo by commercial gene synthesis in 3 kilobase fragments and assembled in Saccharomyces cerevisiae via homologous recombination of 80-basepair overlaps. Subsequent antibiotic selection of cells transfected with the vector resulted in a stable cell line containing the integrated EAA pathway. Finally, we performed a variety of phenotypic, metabolomic, and transcriptomic characterizations on the modified cell line to verify activity of the EAA biosynthesis pathway.

We first confirmed that the CHO cell line was in fact auxotrophic for each of the nine EAAs. As expected, CHO-K1 did not grow in ‘dropout’ F-12K medium lacking each of the nine EAAs and supplemented with dialyzed fetal bovine serum (FBS) (Figure 1—figure supplement 1). We noted that in this cell line, canonically non-essential amino acids tyrosine and proline also exhibited EAA-like properties in dropout media. Insufficient concentrations of phenylalanine in F-12K medium or low expression of endogenous phenylalanine-4-hydroxylase that converts phenylalanine to tyrosine could underlie the tyrosine limitation. Proline auxotrophy in CHO-K1 results from epigenetic silencing of the gene encoding Δ1-pyrroline-5-carboxylate synthetase (P5CS) in the proline pathway (Hefzi et al., 2016). We therefore used proline as a test case for our synthetic genomics pipeline. We tested the P5CS-equivalent proline biosynthesis enzyme found in Escherichia coli, encoded by two separate genes, proA and proB (Figure 1—figure supplement 2A). A vector (pPro) carrying codon-optimized proA and proB separated by a P2A sequence was synthesized and integrated into CHO-K1 (Figure 1—figure supplement 2B). CHO cells with the stably integrated pPro proline pathway showed robust growth in proline-free F-12K medium (Figure 1—figure supplement 2C-D), thus validating a pipeline for designing and generating specific amino acid (AA) prototrophic cells.

To demonstrate restoration of EAA pathways lost from the metazoan lineage more than 650–850 million years ago (Cunningham et al., 2017), we built a 6-gene construct (pMTIV) to investigate the potential rescue of methionine, threonine, isoleucine, and valine auxotrophies. These EAAs were chosen because their biosynthesis pathways were missing the fewest number of genes. Valine and isoleucine collectively require four genes to recapitulate the bacterial-native pathway. (Figure 2—figure supplement 1). The two remaining genes were included to test potential routes to simultaneously rescue threonine and methionine auxotrophy by selectively supplementing individual missing metabolic steps, in addition to complete pathway reconstruction for valine and isoleucine. To biosynthesize methionine, we chose the E. coli metC gene, which encodes cystathionine-ß-lyase and converts cystathionine to homocysteine, a missing step in CHO-K1 cells in a potential serine to methionine biosynthetic pathway. Threonine production was tested using E. coli L-threonine aldolase ltaE, which converts glycine and acetaldehyde into threonine. For branched chain amino acids (BCAAs) valine and isoleucine, three additional biosynthetic enzymes and one regulatory subunit are needed in theory to convert pyruvate and 2-oxobutanoate into valine and isoleucine, respectively. In the case of valine, pyruvate is converted to 2-acetolactate, then to 2,3-dihydroxy-isovalerate, then to 2-oxoisovalerate and finally to valine. For isoleucine, 2-oxobutanoate is converted to 2-aceto-2-hydroxybutanoate, then to 2,3-dihydroxy-3-methylpentanoate, then to 3-methyl-2-oxopentanoate, and finally to isoleucine. The final steps in the biosynthesis of both BCAAs can be performed by native CHO catabolic enzymes Bcat1 and Bcat2 Hefzi et al., 2016. In E. coli, the first three steps in the pathway are embodied in four genes that encode an acetolactate synthase with catalytic and regulatory subunits (ilvB/N), a ketol-acid reductoisomerase (ilvC), and a dihydroxy-acid dehydratase (ilvD) (Figure 2A; Amorim Franco and Blanchard, 2017). The final pMTIV construct comprises metC, itaE, ilvN, ilvB, ilvC, and ilvD organized as a single open reading frame (ORF) with a 2A sequence variant lying between each protein coding region (Figure 2B), and driven by a single strong spleen focus-forming virus (SFFV) promoter.

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Raw cell count data for pMTIV valine-free and complete F-12K medium tests.

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To test the biosynthetic capacity of pMTIV, we first introduced the construct into CHO cells. Flp-In integration was used to stably insert either pMTIV, or a control vector (pCtrl) into the CHO genome. Successful generation of each cell line was confirmed by PCR amplification of junction regions formed during vector integration (Figure 2—figure supplement 2A-B). RNA-seq of cells containing the pMTIV construct confirmed transcription of the entire ORF (Figure 2B). Western blotting of pMTIV cells using antibodies against the P2A peptide yielded bands at the expected masses of P2A-tagged proteins, confirming the production of separate distinct enzymes (Figure 2—figure supplement 2C).

In reconstituted methionine-free, threonine-free, or isoleucine-free F-12K medium supplemented with dialyzed FBS to reduce FBS-derived AA content (Figure 2—figure supplement 3), cells containing the pMTIV construct did not show viability over 7 days, similar to cells containing the pCtrl control vector (Figure 2—figure supplement 4). In striking contrast, however, cells containing the integrated pMTIV showed relatively healthy cell morphology and viability in valine-free F-12K medium (Figure 2C), whereas cells containing pCtrl exhibited substantial loss of viability over 6 days. In complete F-12K medium, cells carrying the integrated pMTIV construct showed no growth defects compared to control cells (Figure 2D). In valine-free F-12K medium, pMTIV cells showed a 32% increase in cell number over 6 days compared to an 88% decrease in cell number in pCtrl cells (Figure 2E). When cultured in valine-free F-12K medium over multiple passages with medium changes every 2 days, pMTIV cell proliferation was substantially reduced by the 3rd passage. We hypothesized that frequent passaging might over-dilute the medium and prevent sufficient accumulation of biosynthesized valine necessary for continued proliferation as has been demonstrated for certain non-essential metabolites which become essential when cells are cultured at low cell densities Eagle and Piez, 1962. We thus deployed a ‘conditioned-medium’ regimen, whereby 50% of the medium was freshly prepared valine-free F-12K medium and 50% was ‘conditioned’ valine-free F-12K medium in which pMTIV cells had previously been cultured over 2 days (see Materials and methods). While use of pMTIV-conditioned medium improved the survival of cells harboring the pathway, it did not completely rescue valine auxotrophy in control cells, which exhibited substantial loss of cell viability over 8 days (Figure 2—figure supplement 5A). As a control, we generated pCtrl-conditioned valine-free F-12K medium using the same medium conditioning regimen, which failed to enable cells to grow to the same degree as that of pMTIV-conditioned medium, suggesting that the benefit conferred by medium conditioning is valine-specific (Figure 2—figure supplement 5B). Using this regimen, we were able to culture pMTIV cells for 9 passages without addition of exogenous valine (Figure 2F). The doubling time was inconsistent across the 49 days of experimentation with cells exhibiting a mean doubling time of 5.3 days in the first 24 days and 21.0 days in the last 25 days. Despite the slowed growth seen in later passages, cells exhibited healthy morphology and continued viability at day-49, suggesting that the cells could have been passaged even further. To verify that the putative valine rescue effect was due to the valine biosynthesis genes present in pMTIV specifically and did not involve confounding effects due to the threonine and methionine biosynthesis genes included in the pMTIV pathway, we constructed and tested a second EAA pathway vector, ‘pIV,’ that only contained the four genes ilvNBCD. The pIV construct similarly supported cell growth in valine-free F-12K medium, and exhibited similar growth dynamics to the pMTIV construct in complete medium (Figure 2—figure supplement 6).

To confirm endogenous biosynthesis of valine, we cultured pCtrl and pMTIV cells in RPMI medium containing ¹³C₆-glucose in the place of its ¹²C equivalent together with ¹³C₃-sodium pyruvate spiked in at 2 mM over three passages (Figure 3—figure supplement 1A). High-resolution MS1 of MTIV cell lysates revealed a peak at 123.1032 m/z, the expected m/z for ¹³C₅-valine (Figure 3A). This detected peak was subject to MS2 alongside a ¹²C-valine control peak and a ¹³C₅/¹⁵N-valine peak, which was spiked into all samples to serve as an internal standard. The resulting fragmentation patterns for each peak (Figure 3B) matched theoretical expectations for each isotopic version of valine (Figure 3—figure supplement 1B). An extracted ion chromatogram further revealed a peak in the pMTIV valine-free medium metabolite extraction, which corresponded to a peak in the spiked-in positive control ¹³C₅/¹⁵N-valine, whereas no equivalent peak was seen among metabolites extracted from pCtrl cells (Figure 3—figure supplement 1C). Taken together, this demonstrates that pMTIV cells are capable of biosynthesizing valine from core metabolites glucose and pyruvate, thereby proving successful metazoan biosynthesis of valine. Over the course of 3 passages in heavy valine-free medium, the non-essential amino acid alanine, which is absent from RPMI medium and synthesized from pyruvate, was found to be 86.1% ¹³C-labeled in pMTIV cell lysates. Assuming similar turnover rates for alanine and valine within the CHO proteome, we expected to see similar percentages of ¹³C-labeled valine. However, just 32.2% of valine in pMTIV cell lysates was ¹³C-labeled (Figure 3—figure supplement 1D-E). For pMTIV cells cultured in heavy but valine-replete medium, just 6.4% of valine in cell lysates was ¹³C-labeled. Together with the observed slow proliferation of pMTIV cells in valine-free medium, our data suggests that valine complementation is sufficient but sub-optimal for cell growth.

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Raw MS/MS data for ¹³C valine labeling experiments.

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We performed RNA-seq to profile the transcriptional responses of cells containing pMTIV or pCtrl in complete (harvested at 0 hr) and valine-free F-12K medium (harvested at 4 hr and 48 hr, respectively) (Figure 3C, Figure 3—figure supplement 2A). The transcriptional impact of pathway integration is modest (Figure 3D). Only 51 transcripts were differentially expressed between pCtrl and pMTIV cells grown in complete medium, and the fold changes between conditions were small (Figure 3E, Figure 3—figure supplement 2B). While some gene ontology (GO) functional categories were enriched (Figure 3—figure supplement 2C), they did not suggest dramatic cellular stress. Rather, these transcriptional changes may reflect cellular response to BCAA dysregulation due to altered valine levels (Zhenyukh et al., 2017), or may result from cryptic effects of bacterial genes placed in a heterologous mammalian cellular context. In contrast, comparison of 48 hr valine-starved pCtrl and pMTIV cells yielded ~7500 differentially expressed genes. Transcriptomes of pMTIV cells in valine-free medium more closely resembled cells grown on complete medium than did pCtrl cells in valine-free medium (Figure 3D, Figure 3—figure supplement 3A). Differentially expressed genes between pCtrl and pMTIV cells showed enrichment for hundreds of GO categories, including clear signatures of cellular stress such as autophagy, changes to endoplasmic reticulum trafficking, and ribosome regulation (Figure 3—figure supplement 3B). Most of the differentially regulated genes between pCtrl cells in complete medium, and those same cells starved of valine for 48 hr were also differentially expressed when comparing pCtrl and pMTIV cells in valine-free medium (Figure 3E), supporting the hypothesis that most of the observed transcriptional changes represent broad but partial rescue of the cellular response to starvation. We also examined the integrated stress response (ISR) and mTOR signaling pathways, both of which are known to modulate cellular responses to starvation (Pakos-Zebrucka et al., 2016). We observed no clear signatures of mTOR activation (Figure 3—figure supplement 4A), although a number of individual genes related to the mTOR pathway were significantly differentially expressed compared to pCtrl cells valine-starved for 48 hr (Figure 3—figure supplement 4B). A manually curated list of ISR genes showed signals of ISR gene activation, but showed few differences between pCtrl and pMTIV cells at 48 hr of starvation (Figure 3—figure supplement 4C). pMTIV cells grown for 5 passages over 29 days on conditioned valine-free F-12K medium were more similar to pMTIV cells starved for 48 hr than to pCtrl cells starved for 48 hr (Figure 3—figure supplement 5A). The transcriptomic signature of these cells, appears slightly orthogonal to the ‘starvation’ effect and recovery of pMTIV and pCtrl cells starved for 48 hr in PCA space (Figure 3—figure supplement 5B), but the majority of the differently expressed genes between pMTIV cells starved for 29 days compared to pCtrl cells grown on complete media are shared with cells starved for 48 hr (Figure 3—figure supplement 5C), suggesting some limited cellular adaptation to long term passaging on valine-free media.

To improve rescue of the valine starvation phenotype, we looked for valine biosynthetic pathway intermediates in our metabolomics data that might suggest that the pathway was bottlenecked at any stage. While no signal could be detected for pyruvate, 2-acetolactate or 2-oxoisovalerate, a signal was detected for pathway intermediate ¹³C₅-2,3-dihydroxy-isovalerate, which was specific to pMTIV cells cultured in both complete and in valine-free medium (Figure 3—figure supplement 1F). To determine whether the downstream pathway gene, ilvD, which encodes the dihydroxy-acid dehydratase enzyme, might constitute a bottleneck, we generated a lentivirus encoding a puromycin resistance cassette in addition to ilvD under control of a viral MMLV promoter (Figure 4A). Both pCtrl and pMTIV cells were infected and integrants were selected for on puromycin, resulting in a population-averaged integration count of 5.7 copies of ilvD for pCtrl cells and 7.1 for pMTIV cells. This resulted in a 0.27- and 0.54-fold increase in ilvD expression (measured as a proportion of uninfected pMTIV ilvD expression) for infected pCtrl (pCtrl-ilvD+) and infected pMTIV cells (pMTIV-ilvD+), respectively (Figure 4B).

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Control and ilvD qPCR data for pMTIV and pMTIV-ilvD + cells, raw cell count data for pMTIV cultured in valine-free RPMI with different BCAA concentrations, and MS/MS data for long-term pMTIV and pMTIV-ilvD+ ¹³C valine labeling experiments.

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We characterized pMTIV and pMTIV-ilvD + cells by culturing these in isotopically heavy valine-free RPMI medium containing ¹³C₆-glucose as before; however, in this experiment 2 mM ¹²C sodium pyruvate was spiked into the medium rather than ¹³C-sodium pyruvate (Figure 4—figure supplement 1A). We also reduced the isoleucine content of this isotopically heavy valine-free RPMI medium to match the isoleucine content of F-12K medium from 0.38 mM to 0.06 mM after observing that pMTIV cells proliferated faster in this condition, presumably due to the high sensitivity of the ilvN/B-encoded acetolactate synthase complex to isoleucine-induced negative feedback inhibition (Barak et al., 1987; Figure 4—figure supplement 1B). pMTIV and pMTIV-ilvD + cells were cultured in this reduced-isoleucine heavy medium over 36 days on plates coated with 0.1% gelatin (Figure 4—figure supplement 1C). We collected duplicate samples at 6 and 10 different timepoints for pMTIV and pMTIV-ilvD+, respectively, throughout the 36 days of culture. Metabolomics analysis revealed ¹³C-valine labeling with most of the valine in both pMTIV and pMTIV-ilvD + cells carrying 0, 2, 3, or 5 ¹³C carbons at both an early (2 days) and a late (24 days) time point in accordance with the expectation when both ¹²C₃ and ¹³C₃-pyruvate (derived from ¹³C_6-glucose) are present (Figure 4C). In pMTIV cells, presence of pathway intermediate 2,3-dihydroxy-isovalerate fluctuated throughout the 24 days of culture with cells exhibiting higher concentrations in earlier time points. In contrast, little or no 2,3-dihydroxy-isovalerate was detected in pMTIV-ilvD + cells across the entire time course, suggesting that the introduction of the extra copies of ilvD is addressing a pathway bottleneck as hypothesized (Figure 4D). In pMTIV-ilvD + cells, the percentage of valine carrying at least one ¹³C-labeled carbon also remained relatively steady throughout the 36 days, while pMTIV cells again exhibited more variability (Figure 4—figure supplement 1D). We calculated the molar content of valine within the cells by relativizing peaks to that of the ¹³C/¹⁵N-valine internal standard, which was spiked into each sample at a known concentration. pMTIV samples on average contained 352.0 picomoles of valine per million cells of which 212.5 picomoles was ¹³C-labeled whereas pMTIV-ilvD + samples on average contained 423.0 picomoles of valine per million cells of which 242.5 picomoles was ¹³C-labeled (Figure 4E). Collectively, the stabilization of 2,3-dihydroxy-isovalerate and valine levels in pMTIV-ilvD + cells suggests that the introduction of additional ilvD-encoded dihydroxy-acid dehydratases is indeed improving flux through the pathway, allowing cells to respond to low valine levels more efficiently compared to pMTIV cells, which results in a more homeostatic cell state.

We quantified the functional impact of modifying flux at this pathway bottleneck by culturing both cell lines in unconditioned, reduced-isoleucine, valine-free RPMI medium containing 2 mM sodium pyruvate over 10 passages on plates not coated with gelatin. When cultured in this medium, pCtrl-ilvD + cells displayed rapid loss of cell viability similar to pCtrl cells (Figure 5—figure supplement 1A). However, pMTIV-ilvD + cells displayed an improved growth phenotype compared to pMTIV cells with an average doubling time of 3.2 days across 39 days and an apparent ability to proliferate indefinitely (Figure 5). By comparison, pMTIV cells exhibited an average doubling time of 4.3 days across 19 days after which they displayed loss of cell viability following passaging events despite both cell lines exhibiting comparable doubling times in complete medium (Figure 5—figure supplement 1B).

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Raw and processed cell count data for pMTIV and pMTIV-ilvD + cultured long-term in unconditioned valine-free RPMI medium.

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In this work, we demonstrated the successful restoration of an EAA biosynthetic pathway in a metazoan cell. Our results indicate that contemporary metazoan biochemistry can support complete biosynthesis of valine, despite millions of years of evolution from its initial loss from the ancestral lineage. Interestingly, independent evidence for BCAA biosynthesis has also been obtained for sap-feeding whitefly bacteriocytes that host bacterial endosymbionts; metabolite sharing between these cells is predicted to lead to biosynthesis of BCAAs that are limiting in their restricted diet. The malleability of mammalian metabolism to accept heterologous core pathways opens up the possibility of animals with designer metabolisms and enhanced capacities to thrive under environmental stress and nutritional starvation (Zhang et al., 2014). Yet, our failure to functionalize designed methionine, threonine, and isoleucine pathways highlights outstanding challenges and future directions for synthetic metabolism engineering in animal cells and animals. Other pathway components or alternative selections may be needed for different EAAs (Rees and Hay, 1995). For instance, the ilvB/N enzyme pair we chose to encode the acetolactate synthase complex in our construct shows feedback sensitivity to isoleucine, which may account for the failure of the pathway to rescue isoleucine auxotrophy. A general lack of predictability and a dearth of well-characterized and controllable genetic ‘parts’ with high dynamic range continue to hamper efforts in genome-scale mammalian engineering (Black et al., 2017; Gilbert et al., 2014; Weingarten-Gabbay et al., 2019). Studies to reincorporate EAAs into the core mammalian metabolism could provide greater understanding of nutrient-starvation in different physiological contexts including the tumor microenvironment (Lim et al., 2020), help answer deep evolutionary questions regarding the formation of the metazoan lineage (Tan et al., 2009), and lead to new model systems or even therapeutics to address metabolic syndrome, Maple Syrup Urine Disease Dancis et al., 1960 and Phenylketonuria (Blau et al., 2010), all of which involve amino acid biosynthetic dysfunction (Kemmer et al., 2010; Ye et al., 2013). Emerging synthetic genomic efforts to build a prototrophic mammal may require reactivation of many more genes (Supplementary files 1-3), iterations of the design, build, test (DBT) cycle, and a larger coordinated research effort to ultimately bring such a project to fruition.

Sequencing data generated for this study is deposited in the NCBI SRA at accession number PRJNA742028. Source data files have been provided for Figure 1 - figure supplement 1, Figure 1 - figure supplement 2D, Figure 2, Figure 2 - figure supplement 3, Figure 2 - figure supplement 4B, Figure 2 - figure supplement 5, Figure 2 - figure supplement 6, Figure 3, and Figure 3 - figure supplement 1, Figure 4, Figure 4 - figure supplement 1, Figure 5, and Figure 5 - figure supplement 1.

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Author details

1. Julie Trolle

   Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Ross M McBee

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2497-3531

2. Ross M McBee

   1. Department of Biological Sciences, Columbia University, New York, United States
   2. Department of Systems Biology, Columbia University, New York, United States

   Present address

   TômTex Inc, Brooklyn, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Julie Trolle

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1926-2863

3. Andrew Kaufman

   Department of Systems Biology, Columbia University, New York, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Sudarshan Pinglay

   Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Henri Berger

   Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States

   Present address

   Weill Cornell Graduate School of Medical Sciences, New York, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Sergei German

   Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

7. Liyuan Liu

   Department of Systems Biology, Columbia University, New York, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. Michael J Shen

   1. Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
   2. Department of Internal Medicine, NYU Langone Health, New York, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

9. Xinyi Guo

   Department of Biology, New York University, New York, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

10. J Andrew Martin

    Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States

    Present address

    Reopen Diagnostics, LLC, Queens, United States

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

11. Michael E Pacold

    Department of Radiation Oncology, NYU Langone Health, New York, United States

    Contribution

    Supervision, Investigation, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3688-2378

12. Drew R Jones

    Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States

    Contribution

    Supervision, Investigation, Methodology

    Competing interests

    No competing interests declared

13. Jef D Boeke

    1. Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    2. Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, United States
    3. Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, United States

    Contribution

    Conceptualization, Supervision, Funding acquisition, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    jef.boeke@nyumc.org

    Competing interests

    is a Founder and Director of CDI Labs, Inc, a Founder of Neochromosome, Inc, a Founder and SAB member of ReOpen Diagnostics, and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc, Modern Meadow, Inc, Rome Therapeutics, Sample6, Inc, Tessera Therapeutics, Inc and the Wyss Institute

    "This ORCID iD identifies the author of this article:" 0000-0001-5322-4946

14. Harris H Wang

    1. Department of Systems Biology, Columbia University, New York, United States
    2. Department of Pathology and Cell Biology, Columbia University, New York, United States

    Contribution

    Conceptualization, Supervision, Funding acquisition, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    hw2429@columbia.edu

    Competing interests

    H.H.W. is a scientific advisor to SNIPR Biome, Arranta Bio, Genus plc, Kingdom Supercultures, Fitibomics, and VecX Biomedicine and a scientific cofounder of Aclid

    "This ORCID iD identifies the author of this article:" 0000-0003-2164-4318

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