Comparative landscape of genetic dependencies in human and chimpanzee stem cells

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 human cells exhibit distinct genetic dependencies. 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 and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.

Keywords: CRISPR screening; G1-phase length hypothesis; cellular anthropology; genetic dependencies; human-specific evolution; neural progenitor cells; pluripotent stem cells.

Figure 1.. Genome-wide CRISPRi screens in human and chimpanzee stem cells identify candidate species-specific genetic dependencies

(A) Schematic of CRISPRi screening approach, with original portraits of Jane Goodall and an adult female chimpanzee from Kibale Chimpanzee Project for artistic representation of human and chimpanzee species differences. 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-v2 sgRNA library, and grown competitively for 10 days. Depleted and enriched sgRNAs were detected by high-throughput sequencing. (B) Scatterplots of sgRNA log₂ fold-change for WTC11 and C3649 technical replicates and UpSet plot showing the intersection of essential genes across all four 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 log₂ fold-change for DepMap essential (bottom left) and nonessential (bottom right) genes. (D) Species-level gene log₂ fold-change across genome-wide CRISPRi screens. Gene-level phenotypes were computed as the mean of the three sgRNAs with the largest absolute log₂ fold-change. sgRNAs lacking perfect-match targets in the chimpanzee genome were excluded from analysis. See also Figure S1 and Tables S1-S3.

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 t_final (red and blue circles). (D) Scatterplot of log₂ fold-change of sgRNA counts, modeled 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 log₂ fold-change across validation CRISPRi screens. Gene-level phenotypes were computed as the mean of the four sgRNAs with the largest absolute log₂ fold-change. The 12 genes with the greatest variance in sgRNA log₂ 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. t_final). (G) Heatmap of gene log₂ fold-change and hierarchical clustering for 75 genes with species-specific effects on cellular proliferation across validation CRISPRi screens (1% FDR). See also Figures S2, S3, and Tables S4-S5.

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 log₂ fold-change for sgRNAs targeting ATP6AP1, ATP6AP2 and ATP6V0C. Data derived from CEV-v1 validation screens for ATP6AP1, ATP6AP2 and only from primary genome-wide screen plotted for ATP6V0C. (C) Strip plots of log₂ fold-change for sgRNAs targeting CDK2, CCNE1, and CDK4. Each circle represents the sgRNA log₂ 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) qRT-PCR measurements of knockdown efficiency in human (28126B, blue, n = 3) and chimpanzee (40280L, red, n = 3) PSCs. (E) Western blot for phospho-S6 (pS6) expression and GAPDH loading control for three wild-type human (H1, 21792A, and 28126B) and three wild-type chimpanzee (3624K, 40280L, and 8861G) cell lines, and cell lines depleted for ATP6AP1 or ATP6AP2 (28126B and 40280L). See also Figures S4.

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 human (28126B, blue) and chimpanzee (40280L, red) cells in G1 phase upon knockdown of CDK2 (P < 10⁻³, n = 2), Cyclin E1 (P < 10⁻³, n = 2), RBL1 (P < 10⁻², n = 6), and FAM122A (P < 0.05, n = 2), calculated by two-tailed t-test. (D) Comparative gene expression data from human and chimpanzee PSCs for core cell cycle regulators (* P < 0.05, ** P < 10⁻², *** P < 10⁻³, P-values calculated in Gallego Romero et al., 2015). (E) qRT-PCR measurements of the degree of CDK1 overexpression (n = 2 with two qRT-PCR primer sets, Table S3). (F) Change in the fraction of cells in G1 phase upon overexpression of CDK1 in conjunction with CDK2 or Cyclin E1 knockdown. (G) Change in the fraction of FAM122A sgRNA containing cells in the presence of no drug, C1 inhibitor prexasertib (CHK1i), or WEE1 inhibitor adavosertib (WEE1i) (n = 2, two days of drug treatment). (H) Fraction of wild-type human (blue, 21792A) vs. wild-type chimpanzee (red, 40280L) cells grown in co-culture in the presence of no drug CHK1i, or WEE1i (n = 4). For all bar charts, data are plotted as mean ± s.e.m with individual data points representing biological replicates. See also Figure S5, and Video S1.

Figure 5.. Human-specific robustness to cell cycle perturbations persist 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 human (28126B, blue) and chimpanzee (40280L, red) NPCs in G1 phase upon depletion of CDK2 (P < 0.05, N=4), Cyclin E1 (P < 10⁻³, n = 4 and n = 3), or RBL1 (n.s., n = 2), calculated by two-tailed t-test. (D) Change in the fraction of NPCs in G2 phase upon depletion of FAM122A (P < 0.05, two-tailed t-test, n = 2 and n = 1). Bar charts in (C) and (D) plotted as mean ± s.e.m, with individual data points representing biological replicates. See also Figure S6.

Figure 6.. Human robustness to cell cycle perturbations in cerebral organoids

(A) Organoid size measurements for human (H1) and chimpanzee (8861G) cerebral organoids, measured on day 18 by brightfield microscopy (n = 4 to 12). Bar charts plotted as mean ± s.e.m, with each individual data point representing an independent organoid. (B) Representative images of organoids from each sgRNA condition and corresponding image segmentation. (C) Cell cycle measurements for day 9 human (H1, 20961B, and 23555A; n = 2, n = 1, n = 1; blue) and chimpanzee (8861G and 40280L; n = 2 and n = 1; red) organoids. Change in the fraction of cells in G1 phase in cells expressing an sgRNA targeting CDK2 (P < 0.001, two-tailed t-test) or a non-targeting sgRNA (P = 0.56, two-tailed t-test).

Figure 7.. Orangutan PSCs suggest evolutionary origin of species-specific genetic dependencies

(A) Change in the relative fraction of CDK2 sgRNA containing cells over time in human (blue, 28126B), chimpanzee (red, 40280L), and orangutan PSCs (purple, n = 3 for each species) (** P < 10⁻², *** P < 10⁻³, P-values calculated by two-tailed t-test on final timepoint). qRT-PCR measurements of sgRNA knockdown efficiency for each sgRNA in all three species (n = 1 to 3). (B-E) Relative sgRNA fraction over time and qRT-PCR measurements for sgRNAs targeting (B) CDK4, (C) ATP6AP1, (D) KAT6A, and (E) UFL1. Individual data points represent biological replicates and qRT-PCR bar charts plotted as mean ± s.e.m. See also Figure S7.