Abstract
Systemic characterization of genes and pathways underlying the genetic architecture of type 2 diabetes (T2D) requires scalable functional genomics approaches. Molecular readouts from CRISPR perturbations can effectively uncover the mechanistic effects of underexplored genes. Here we performed single-cell RNA sequencing on pooled CRISPR screens (Perturb-seq) of 61 T2D-associated genes and 40 ribosome-associated quality control (RQC) genes in human pancreatic β cells (EndoC-βH1) for investigations of insulin production and T2D pathology. We identified 21 functional genes, including the uncharacterized KLHL42 and ZZEF1. Findings from global and β cell-specific knockout male mice, islet organoids and human islets reveal that ZZEF1 is a regulator of insulin synthesis and β cell stress through ribosomal stress-surveillance pathways in working and stress status-defined β cell subtypes. ZZEF1 deficiency impairs β cell function by inhibiting the RQC sensor EDF1, which could be improved by azoramide and ISRIB treatments. These Perturb-seq analyses and identification of functional RQC-related genes can provide potential therapeutic targets for T2D.
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Data availability
The mass spectrometry proteomics data (accession number PXD054785) have been deposited in the PRIDE database. All raw sequencing data, including Endoc-βH1 cell line Perturb-seq screens (accession number GSE273677), mouse scRNA-seq (accession numbers GSE273688 and GSE288211) and mouse bulk RNA-seq (accession numbers GSE223143 and GSE288210), have been deposited in the Gene Expression Omnibus. The public datasets used in this study include human RNA-seq datasets (accession numbers GSE50244, GSE159984 and GSE164416), oligonucleotide microarray data (accession number GSE76896), scRNA-seq datasets (accession numbers GSE128565 and GSE162512) and proteomics datasets of human islets from Human Islets Consortium (https://www.humanislets.com/#/)65. Raw images are included in the Source data. The histology images have been deposited in Zenodo (https://doi.org/10.5281/zenodo.17163230)66. Source data are provided with this paper.
Code availability
This study was conducted using publicly available software. No custom code was used.
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Acknowledgements
Y.C. acknowledges support from the Natural Science Foundation of China (82270842, 82470830), Noncommunicable Chronic Diseases—National Science and Technology Major Project (2023ZD0507700, 2023ZD0507702) and the National Key Research and Development Program of China (2020YFA0112800). X.L. is supported by the National Postdoctoral Program for Innovative Talents (BX20220210). The computations in this work were run on the π 2.0 cluster supported by the Center for High Performance Computing, and the liquid chromatography–mass spectrometry proteomics analyses were supported by the Instrumental Analysis Center at Shanghai Jiao Tong University. We appreciate Y. A. Zeng (Chinese Academy of Sciences, Shanghai) for collaboration on human islet isolation. All schematic figures were created using BioRender.com.
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Contributions
Y.C. conceived the project, contributed to research data and data analysis. J.N., X.L., D.L., Y.S., A.S. and X.J. contributed to the mouse model and to cellular and molecular experiments. J.N. and X.L. contributed to RNA sequencing and data collection. X.H., J.R., J.C., P.L. and Y.C. performed the bioinformatics analysis. J.X. contributed to the human sample analyses. G.N. and W.W. contributed to the discussion of the results. J.N. and Y.C. wrote the paper; Y.C. revised the paper.
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Nature Metabolism thanks Jean-Sébastien Annicotte, Adolfo Garcia-Ocana and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Revati Dewal, in collaboration with the Nature Metabolism team.
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Extended data
Extended Data Fig. 1 Quality control and analysis for Perturb-seq screening of T2D susceptibility genes.
a, Cell proportion of detected sgRNA for each targeting gene. b, Cell proportion of 7 replicate Perturb-seq data for each targeting gene. c, Cell proportion of 7 EndoC-βH1 clusters for each targeting gene. d, The number of perturbed cells for each targeting gene and non-targeting control. The gray dashed line represents a filtering threshold of 50 cells. e, The enriched GO terms of perturbation effects of CALCOCO2, SLC30A8, SND1 and THADA in EndoC-βH1 cells. P values were calculated by one-sided hypergeometric test. BP, biological process.
Extended Data Fig. 2 mRNA and protein expression of ZZEF1 in human islets.
a, Normalized expression levels of ZZEF1 (FPKM from RNA-seq or log2 RMA signal intensity from oligo microarray) in human islets from non-diabetic (ND), impaired glucose tolerance (IGT) and T2D individuals. PPP, partially pancreatectomized patient. OD, organ donor. RMA, robust multi-array average. P values were calculated by two-sided Wilcoxon test (GSE50244 n = 52, 15, 10; GSE159984 n = 58, 27; GSE164416 n = 18, 38, 39) or the empirical Bayes moderated two-sided t-test in limma package (GSE76896 PPP group n = 32, 15, 36; OD group n = 84, 19). b, Relative protein levels of ZZEF1 (log2 normalized abundance from bulk proteomics) in human islets from male and female ND and T2D individuals (n = 77, 40, 10, 7). P values were calculated using the empirical Bayes moderated two-sided t-test in limma package. Boxplots display the median (center line), interquartile range (IQR, box edges), whiskers extending to the most extreme values within 1.5 × IQR. Data points past the whiskers are considered outliers and are plotted individually. c,d, Representative immunohistochemical staining of ZZEF1 on the pancreatic sections from male and female ND and T2D individuals. Scale bars, 50 μm (c). H-scores were analyzed in islets of different sections (n = 8, 8, 7, 9) (d). e-h, Representative IF staining of ZZEF1 (green), C-pepteide (red) and DAPI (blue) on pancreatic sections from male and female ND and T2D individuals. Scale bars, 50 μm (e). The mean fluorescence intensity of ZZEF1 was analyzed (n = 8, 8, 5, 6) (f). The proportions of ZZEF1+ cells in islets (g) and ZZEF1+C-peptide+ cells in C-peptide+ cells (h) were analyzed (n = 8, 8, 5, 6). The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D. P values were calculated by unpaired two-tailed Student’s t-test (2 groups).
Extended Data Fig. 3 Generation and characterization of β-cell-specific Zzef1-KO mice.
a, Schematic illustration of the generation of Zzef1flox/flox mice, βZKO-MIP and βZKO-RIP mice. The Zzef1f/f mice were generated by selecting exon 15 of Zzef1 as the conditional knockout region. The blue arrows showed the design of primers for genotyping. b, DNA genotyping of Zzef1 conditional knockout mice. c, Representative IHC staining of ZZEF1 on the pancreatic sections of βZKO-MIP, βZKO-RIP mice and control mice. Scale bars, 50 μm. d, The mRNA expression level of Zzef1 in the isolated islets from βZKO-MIP, βZKO-RIP and control mice (n = 5 for each group). e-g, Random blood glucose (n = 14, 17, 17, 14, 17, 17) (e), body growth curves (n = 12, 14, 15, 10, 11, 12) (f), body weight, fat mass and lean mass (n = 6, 7, 8, 7, 8, 8) (g) of βZKO-MIP and βZKO-RIP mice fed with HFD. h, Oxygen consumption (VO2), carbon dioxide generation (VCO2), respiratory exchange ratio (RER) and heat production of HFD-fed βZKO-MIP (n = 8 for each group) and βZKO-RIP mice (n = 8, 8; n = 8, 8; n = 8, 8; n = 7, 8). i,j, IPGTT of βZKO-MIP (n = 5, 8, 6, 6) (i), βZKO-RIP (n = 6, 7, 6, 5) (j), littermate controls and wild-type control mice fed with NCD. The statistical significance of AUC differences was determined. k, Quantification of pancreatic insulin content in βZKO-MIP, βZKO-RIP and control mice fed with NCD (n = 4 for each group). l-o, Western blot (l, n) and quantification (m, o) of ZZEF1 and insulin protein levels in islets from HFD-fed βZKO-MIP, βZKO-RIP and control mice (n = 3 for each group). All mice were male. The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D. P values were calculated by unpaired two-tailed Student’s t-test (2 groups) or one-way ANOVA followed by Tukey’s test (4 groups).
Extended Data Fig. 4 Inhibition and activation of ZZEF1 expression in isolated human islets.
a, Representative images of human islets transfected with ZZEF1-knockdown (KD-shZZEF1) or non-targeting control (Ctrl-shNT) GFP-expressing adenovirus. Scale bars, 20 μm. b,c, Western blots (b) and quantification analysis (c) of ZZEF1 in the adenovirus-transfected human islets (n = 3 for each group). d, Quantification of insulin content in the adenovirus-transfected human islets (n = 3 for each group). e, Representative images of human islets transfected with CRISPR activation of ZZEF1 (CRISPRa-ZZEF1) or non-targeting control (CRISPRa-NT) by a dual adenoviral system (GFP-expressing adenovirus with sgRNA and mCherry-expressing adenovirus with dCas9). Scale bars, 20 μm. f,g, Western blots (f) and quantification analysis (g) of ZZEF1 in CRISPRa and control adenovirus-transfected human islets (n = 3 for each group). h, Quantification of insulin content in CRISPRa and control adenovirus-transfected human islets (n = 3 for each group). i,j, Western blots (i) and quantification analysis (j) of ZZEF1 in the knockdown (KD-shZZEF1) and non-targeting control (Ctrl-shNT) lentivirus-transfected βH1 cells (n = 3 for each group). k, GSIS tests for the βH1 cells transfected with ZZEF1-KD or control lentivirus (k). The normalized SI was measured (n = 4, 3). l, Quantification of insulin content in ZZEF1-KD and control lentivirus-transfected βH1 cells (n = 4 for each group). The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D. P values were calculated by unpaired two-tailed Student’s t-test (2 groups) or two-way ANOVA followed by Bonferroni’s post-hoc test for multiple comparisons (GSIS).
Extended Data Fig. 5 Generation and characterization of global Zzef1 knockout mice.
a, Schematic illustration of the generation of global Zzef1 knockout mice (Zzef1−/−) by CRISPR /Cas9-mediated genome editing of exon 2-3. The blue arrows showed the design of primers for genotyping. b, DNA genotyping of Zzef1 knockout and control mice (homozygous Zzef1−/−, heterozygous Zzef1+/−, wild-type Zzef1+/+). c, The mRNA expression levels of Zzef1 in tissues from NCD-fed Zzef1−/− and Zzef1+/+ mice (n = 7, 8, 4, 4, 4, 4, 4, 4, 8, 8, 4, 4, 8, 8, 4, 4, 4, 4). d, Representative IHC staining of ZZEF1 on the pancreatic sections of HFD-fed Zzef1−/− and Zzef1+/+ mice. Scale bars, 50 μm. e-g, Random blood glucose (n = 17 for each group) (e), body growth curves (n = 15, 15, 17, 16) (f) and body fat mass, lean mass, and body weight (n = 8, 9) (g) of Zzef1−/− and Zzef1+/+ mice. h,i, IPGTT (n = 10, 13, 17, 19) (h) and IRT (n = 12, 12, 5, 7) (i) of Zzef1−/− and Zzef1+/+ mice. The statistical significance of AUC differences was determined. j, GSIS tests for the islets from Zzef1−/− and Zzef1+/+ mice fed with NCD or HFD. The normalized SI was measured (n = 7, 4, 4, 5). k, Quantification of pancreatic insulin content of Zzef1−/− and Zzef1+/+ mice (n = 3, 4, 3, 4). l, Quantification of β-cell mass of HFD-fed Zzef1−/− and Zzef1+/+ mice (n = 3 for each group). m, Proportions of α, β, δ, PP cells and insulin+glucagon+ (INS+GCG+) in the flow cytometry analysis of islets from HFD-fed Zzef1−/− and Zzef1+/+ mice (n = 3 for each group). n,o, Representative transmission electron microscopy (n) and quantitative analysis (o) of vesicles containing mature (red) and immature (blue) insulin-dense core granules in the β-cells of Zzef1−/− and Zzef1+/+ mice fed with HFD (n = 4, 5). Scale bars, 1 μm. p, VO2, VCO2, RER and heat production of 20-week-old Zzef1−/− (n = 7) and Zzef1+/+ (n = 8) mice fed with NCD. All mice were male. The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D. P values s were calculated by unpaired two-tailed Student’s t-test (2 groups), two-way ANOVA followed by Bonferroni’s post-hoc test for multiple comparisons (GSIS), one-way ANOVA followed by Tukey’s test (4 groups).
Extended Data Fig. 6 scRNA-seq analysis of islet cells in βZKO-MIP and βZKO-RIP mice.
a, The UMAP of islet cells from βZKO-MIP and control mice fed with NCD or HFD (left, 46,393 cells), βZKO-RIP and control mice fed with NCD or HFD (right, 44,242 cells). Each group consists of two replicates; each replicate contains islets from at least three mice. b, Proportion of cell types in the islets from βZKO-MIP, βZKO-RIP and control mice fed with NCD or HFD. Alpha, α-cells; Beta, β-cells; Delta, δ-cells; PP, pancreatic polypeptide cells. c,d, Bubble plot of marker genes expression in the islet cell types of βZKO-MIP (c), βZKO-RIP (d) and control mice. e,f, Proportions of α, β, δ, PP cells and insulin+glucagon+ (INS+GCG+) in the flow cytometry analysis of islets from HFD-fed βZKO-MIP (e), βZKO-RIP (f) and control mice (n = 3 for each group). P values were calculated by unpaired two-tailed Student’s t-test. g, The separated UMAP of insulin-expressing cells based on scRNA-seq of islets from NCD-fed 12-week-old βZKO-MIP, βZKO-RIP and control mice, and HFD-fed 20-week-old βZKO-MIP, βZKO-RIP and control mice. h, Proportion of 4 β-cell subtypes in the islets from βZKO-MIP, βZKO-RIP and control mice fed with NCD or HFD. i, Bubble plot of representative marker genes for the β-cell subtypes of βZKO-MIP, βZKO-RIP and control mice fed with NCD or HFD. j, Enriched GO terms of common DEGs between Zzef1-KO and control β-cells from βZKO-MIP, βZKO-RIP mice fed with NCD or HFD. P values were calculated by one-sided hypergeometric test. k, Violin plots of Ins1 and Ins2 expression in β-cells of βZKO-MIP, βZKO-RIP and control mice fed with NCD or HFD (βZKO-MIP n = 7,160, 9,436, 9,855, 7,370; βZKO-RIP n = 6,495, 5,578, 7,420, 11,942). P values were calculated by two-sided Wilcoxon rank-sum test. Boxplots display the median (center line), IQR (box edges), whiskers extending to the most extreme values within 1.5 × IQR. Data points beyond the whiskers are considered outliers and are plotted individually. All mice were male. The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D.
Extended Data Fig. 7 ZZEF1 regulates the expression of Edf1.
a, Representative staining of insulin (red), direct fluorescence of AAV8-coexpressed GFP (green) and DAPI (blue) in the islets of mice with pancreatic infusion of AAV8-MIP-EDF1. Scale bars, 20 μm. b, Dual-luciferase reporter assay for Edf1 in the human ZZEF1-overexpressed HEK293T cells or mouse Zzef1-overexpressed βTC6 cells (n = 3 for each group). c, ChIP-qPCR analysis of the activity of Edf1 promoter regions in the ZZEF1-overexpressed HEK293T cells (n = 2 for each group). d, The expression levels of Edf1 in different tissues from Zzef1−/− and Zzef1+/+ mice fed with NCD (n = 7, 8, 5, 5, 6, 6, 5, 5, 8, 8, 8, 8, 6, 5). e,f, Western blots and quantification analysis of ZZEF1, EDF1, G3BP1, T-eIF2α and P-eIF2α in the hypothalamus (e) and fat (f) of NCD-fed Zzef1−/− and Zzef1+/+ mice (n = 4 for each group). All mice were male. The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D. P values were calculated by unpaired two-tailed Student’s t-test.
Extended Data Fig. 8 Azoramide treatment restores β-cell function in mice.
a, Schematic schedule for azoramide treatments and experiments in mice. b,c, Body weights of the βZKO-MIP and control mice treated with azoramide or vehicle (n = 5, 6, 7, 6) (b), or ISRIB or vehicle (n = 5, 6, 6, 6) (c). d, Representative RNAscope FISH of hormone genes (Gcg, Ins2, Ppy, Sst) on the pancreatic sections of βZKO-MIP and littermates control mice treated with azoramide or vehicle. Scale bars, 20 μm. e,f, IF staining of insulin (white), cocktail of GCG/SST/PPY (red), CHGA (green) and DAPI (blue) on pancreatic sections of HFD-fed βZKO-MIP and control mice (e). The yellow arrows point at the CHGA+ hormone− dedifferentiated cells, and their proportion in islets was analyzed (n = 3 for each group) (f). Scale bars, 20 μm. All mice were male. The number of samples per group is indicated at the bottom of bars in the graphs. Data represented as mean ± S.D. P values were calculated by unpaired two-tailed Student’s t-test.
Extended Data Fig. 9 Quality control and analysis for Perturb-seq screening of RQC genes.
a, Cell proportion of detected gRNA for each targeting gene. b, The proportion of perturbation reads for each targeting gene in the 2 replicate Perturb-seq data. c, Cell proportion of perturbed βH1 cell clusters for each targeting gene. d, UMAP of the perturbed cells and control cells in the βH1 cell clusters. e, UMAP of βH1 cell clusters in the 2 replicate Perturb-seq data.
Extended Data Fig. 10 Schematic illustration of mechanistic role of ZZEF1 and experimental workflow.
a, Schematic illustration of ZZEF1 functions in pancreatic β-cells. b, Illustration of experimental workflow.
Supplementary information
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Nan, J., He, X., Liu, X. et al. Single-cell perturbations decipher ribosomal stress-surveillance regulators in type 2 diabetes. Nat Metab 8, 139–158 (2026). https://doi.org/10.1038/s42255-025-01407-6
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DOI: https://doi.org/10.1038/s42255-025-01407-6
