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[Preprint]. 2023 Mar 20:2023.03.19.533346.
doi: 10.1101/2023.03.19.533346.

Comparative landscape of genetic dependencies in human and chimpanzee stem cells

Richard She  1   2 , Tyler Fair  3   4   2 , Nathan K Schaefer  3   5 , Reuben A Saunders  1   6 , Bryan J Pavlovic  3   5 , Jonathan S Weissman  1   7   8   9 , Alex A Pollen  3   5   10
Affiliations

Comparative landscape of genetic dependencies in human and chimpanzee stem cells

Richard She et al. bioRxiv. .

Update in

Abstract

Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether changes in human cells alter requirements for essential genes. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells, providing support for the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells can reshape the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.

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Conflict of interest statement

Declaration of interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Genome-wide CRISPRi screens in human and chimpanzee stem cells identify candidate species-specific genetic dependencies
(A) Schematic of CRISPRi screening approach. Two human (WTC11 and H1) and two chimpanzee (C3649 and Pt5-C) PSC lines were engineered to express dCas9-KRAB, infected with the lentiviral hCRISPRi-2 sgRNA library, and grown competitively for 10 days. Depleted and enriched sgRNAs were detected by high-throughput sequencing. (B) Scatterplots of sgRNA fold-change for WTC11 and C3649 technical replicates and UpSet plot showing the intersection of essential genes across screens. (C) Precision-recall analysis (top left) for each screen. Precision and recall were determined using DepMap essential and nonessential genes. The number of DepMap essential genes (top right) identified by MAGeCK (5% FDR, log2 fold-change < −1.5). Distribution of fold-change for DepMap essential (bottom left) and nonessential (bottom right) genes. (D) Species-level gene fold-change across genome-wide CRISPRi screens. Gene-level phenotypes were computed as the mean of the three sgRNAs with the largest absolute fold-change. sgRNAs lacking perfect-match targets in the chimpanzee genome were excluded from analysis.
Figure 2.
Figure 2.. Species-specific genetic dependencies validate across five human and six chimpanzee individuals
(A) Schematic of validation sgRNA library design and CRISPRi screening approach. (B) Heatmap of Pearson correlations and hierarchical clustering for sgRNA profiles across 16 validation CRISPRi screens. Individuals listed twice are replicate screens performed in separate laboratories. (C) Principal component analysis of sgRNA counts at t0 (black circle) and tfinal (red and blue circles). (D) Scatterplot of log2 fold-change of sgRNA counts, weighted and normalized by DESeq2. 1,133 sgRNAs with significant species differences (FDR < 0.01) colored in purple and negative-control sgRNAs colored in dark gray. (E) Species-level gene fold-change across validation CRISPRi screens. Gene-level phenotypes were computed as the mean of the four sgRNAs with the largest absolute fold-change. The 12 genes with the greatest variance in sgRNA fold-change attributable to species are labeled. (F) Dream-variancePartition analysis for quantifying sources of variation in sgRNA counts attributable to individual, species, and timepoint (t0 vs. tfinal). (G) Heatmap of gene fold-change and hierarchical clustering for 75 genes with species-specific effects on cellular proliferation across validation CRISPRi screens (1% FDR).
Figure 3.
Figure 3.. Core species-specific genetic dependencies
(A) Species-specific genetic dependencies with STRING protein-protein associations. Illustrations of pathways and protein complexes with coherent species-specific effects. (B) Strip plots of fold-change for sgRNAs targeting ATP6AP1, ATP6AP2 and ATP6V0C (C) CDK2, CCNE1, and CDK4. Each circle represents the sgRNA fold-change for one sgRNA in one human (blue) or chimpanzee (red) individual. Each stripplot contains a variable number of columns, corresponding to the number of significant sgRNAs targeting each gene. (D) Heatmap of RNA-seq expression data for human (WTC11) and chimpanzee (C3649) cells depleted for KAT6A or BRPF1. (E) Western blot for phospho-S6 expression and GAPDH loading control for three wild-type human (H1, 21792A, and 28128B) and three wild-type chimpanzee (3624K, 40280L, and 8861G) cell lines, and cell lines depleted for ATP6AP1 or ATP6AP2 (28128B and 40280L).
Figure 4.
Figure 4.. Divergent regulation of cell cycle progression in human and chimpanzee cells
(A) Schematic for Cdk1/Cdk2 regulatory network. Cdk1 and Cdk2 phosphorylate key substrates Wee1 and Cdc25, leading to degradation of Wee1 and activation of Cdc25. Phosphatase PP2A dephosphorylates Wee1 and Cdc25 at the same sites. Chk1 inhibits FAM122A, and FAM122A inhibits PP2A. (B) Cell cycle proportions in chimpanzee wild-type cells (GFP−) and sgRNA containing cells (GFP+) grown in co-culture. (C) Change in the fraction of cells in G1 phase upon knockdown of CDK2 (P < 10−3), Cyclin E1 (P < 10−3), RBL1 (P < 10−2), and FAM122A (P < 0.05). (D) qRT-PCR measurements of knockdown efficiency in human (blue) and chimpanzee (red) PSCs. (E) Comparative gene expression data from human and chimpanzee PSCs for core cell cycle regulators (* P < 0.05, ** P < 10−2, *** P < 10−3). (F) Change in the fraction of cells in G1 phase upon overexpression of CDK1 in conjunction with CDK2 or Cyclin E1 knockdown. (G) qRT-PCR measurements of the degree of CDK1 overexpression. (H) Change in the fraction of FAM122A sgRNA containing cells in the presence of no drug or Chk1 inhibitor prexasertib (Chk1i). (I) Fraction of wild-type human (blue) vs. wild-type chimpanzee (red) cells grown in co-culture in the presence of no drug or Chk1i.
Figure 5.
Figure 5.. Differentiation into neural progenitor cells
(A) Schematic for differentiation of PSCs into neural progenitor cells (NPCs). (B) Cell cycle proportions in human wild-type neural progenitor cells (GFP−) and sgRNA containing cells (GFP+) grown in co-culture. (C) Change in the fraction of NPCs in G1 phase upon depletion of CDK2 (P < 0.05), Cyclin E1 (P < 10−3), or RBL1 (n.s.). (D) Change in the fraction of NPCs in G2 phase upon depletion of FAM122A (P < 0.05).
Figure 6.
Figure 6.. Orangutan PSCs reveal evolutionary origin of species-specific genetic dependencies
(A) Change in the relative fraction of CDK2 sgRNA containing cells over time in human, chimpanzee, and orangutan PSCs. qRT-PCR measurements of sgRNA knockdown efficiency for each sgRNA in all three species. (B-E) Relative sgRNA fraction over time and qRT-PCR measurements for sgRNAs targeting (B) CDK4, (C) ATP6AP1, (D) KAT6A, and (E) UFL1.
Figure 7.
Figure 7.. Model for inputs to neural progenitor differentiation
(A) Schematic for changes in cell cycle length and proportion in pluripotent stem cells and neural progenitor cells with intrinsic and extrinsic inputs that can potentially influence the duration of G1 phase.
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References

    1. Adamson B., Norman T.M., Jost M., Cho M.Y., Nuñez J.K., Chen Y., Villalta J.E., Gilbert L.A., Horlbeck M.A., Hein M.Y., et al. (2016). A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. Cell 167, 1867–1882.e21. 10.1016/j.cell.2016.11.048. - DOI - PMC - PubMed
    1. Aldea D., Atsuta Y., Kokalari B., Schaffner S.F., Prasasya R.D., Aharoni A., Dingwall H.L., Warder B., and Kamberov Y.G. (2021). Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution. Proc. Natl. Acad. Sci. 118, e2021722118. 10.1073/pnas.2021722118. - DOI - PMC - PubMed
    1. Aleem E., Kiyokawa H., and Kaldis P. (2005). Cdc2-cyclin E complexes regulate the G1/S phase transition. Nat. Cell Biol. 7, 831–836. 10.1038/ncb1284. - DOI - PubMed
    1. Baker D.E.C., Harrison N.J., Maltby E., Smith K., Moore H.D., Shaw P.J., Heath P.R., Holden H., and Andrews P.W. (2007). Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25, 207–215. 10.1038/nbt1285. - DOI - PubMed
    1. Becker K.A., Ghule P.N., Therrien J.A., Lian J.B., Stein J.L., van Wijnen A.J., and Stein G.S. (2006). Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J. Cell. Physiol. 209, 883–893. 10.1002/jcp.20776. - DOI - PubMed

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