Zheng Hu

BACKGROUND: Cervical cancer (CC) remains the fourth leading cause of cancer-related deaths in women globally, with poor prognosis for metastatic and recurrent cases. Although genomic alterations have been extensively characterized, global proteogenomic landscape of the disease is largely under explored. METHODS: Here, we present the first genome-wide proteogenomic characterization of CC, analyzing 139 tumor-normal tissue pairs using whole-genome sequencing, transcriptomics, proteomics, and phosphoproteomics. RESULTS: We identified 4 distinct molecular subtypes with unique clinical outcomes: epithelial-mesenchymal transition (EMT, C1), proliferation (C2), immune response (C3), and epithelial differentiation (C4). A 4-protein classifier (CDH13, TP53BP1, NNMT, HSPB1) was developed with strong prognostic and predictive value, particularly for immunotherapy response in subtype C3. Phosphoproteomic profiling uncovered subtype-specific kinase activity, identifying actionable therapeutic targets. CONCLUSION: Our findings further revealed previously uncharacterized somatic copy number alterations, extrachromosomal DNA landscape, and human-HPV fusion peptides, with implications for genetic heterogeneity and therapeutic targets. This study enhances the understanding of cervical cancer through deeper proteogenomic insights and facilitates the development of personalized therapeutic strategies to improve patient outcomes. FUNDING: Noncommunicable Chronic Diseases-National Science and Technology Major Project (2025ZD0544102);The National Natural Science Foundation of China (82172584); Key Technology R&D Program of Hubei (2024BCB057 and 2025BCB053); National Natural Science Foundation of China (82373260); the “4+X” clinical trial programs of Women’s Hospital, School of Medicine, Zhejiang University (LY2022004); and the programs of Zhejiang Traditional Chinese Medicine Innovation Team (CZ2024009); and Guangxi Natural Science Foundation (2024GXNSFBA010045).

Objective: To compare the diagnostic accuracy of minipad collected menstrual blood versus clinician collected cervical samples to test for human papillomavirus (HPV) in the detection of cervical intraepithelial neoplasia grade 2/3 or worse (CIN2+/CIN3+). Design: Cross sectional population based study. Setting: Four urban and three rural communities in Hubei Province, China. Participants: 3068 women aged 20-54 years with regular menstrual cycles, enrolled between September 2021 and January 2025. Interventions: HPV testing using minipad collected menstrual blood, clinician collected cervical samples, and ThinPrep cytology. Women who tested HPV positive by either collection method or by cytology (atypical squamous cells of undetermined significance or worse) were referred for colposcopy directed biopsy sampling. Main outcome measure: Diagnostic accuracy for detecting CIN2+ and CIN3+. Results: Among 3068 participants, minipad based HPV testing showed a sensitivity of 94.7% (95% confidence interval 80.9% to 99.1%) for CIN2+ detection, comparable to clinician based HPV testing (92.1%, 77.5% to 97.9%; P=1.00). Although minipad HPV testing showed a lower specificity than clinician HPV testing (89.1%, 88.0% to 90.2% v 90.0%, 88.9% to 91.1%; P=0.001), the negative predictive value matched that of clinician HPV testing (99.9%, 99.7% to 100.0% v 99.9%, 99.7% to 100.0%; P=1.00). Both collection methods had a similar positive predictive value (9.9%, 7.1% to 13.5% v 10.4%, 7.4% to 14.3%; P=0.82) and screening efficiency (10.1 v 9.6 referrals per CIN2+ detected; P=0.82). Conclusions: Minipad collected menstrual blood showed comparable diagnostic accuracy to clinician collected cervical samples for HPV testing for detecting CIN2+ and CIN3+. Trial registration: ClinicalTrials.gov NCT06082765 .

DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase’s spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE’s versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.

High‐risk human papillomavirus (hrHPV) infection is the primary cause of cervical cancer. However, hrHPV testing lacks specificity in detecting neoplastic changes. This study explored the utility of quantitative methylated HPV DNA markers for precise detection of cervical lesions. Using hybridization capture‐based bisulfite sequencing, we analyzed genome‐wide HPV methylation patterns. The study included a training cohort of 60 cervical exfoliated cell samples and a validation cohort of 29 samples. Analysis of 112 CpG sites across the HPV genome revealed that genome‐wide HPV16 methylation levels correlated with disease progression. Squamous cell carcinoma (SCC) showed 1.4‐fold higher methylation levels compared to normal tissue ( p = 0.0032). Progressive methylation increases in the E5‐α and L2 genes were observed across the spectrum of cervical lesion severity, from normal tissue through high‐grade squamous intraepithelial lesion (HSIL) to SCC. Intersection analysis of differentially methylated CpG sites between HSIL vs Normal and SCC vs Normal identified 16 consistently hypermethylated CpG sites in the E5‐α, E7, L2, and L1 genes, distinguishing both HSIL and SCC from normal tissue. This pilot study identifies a five‐CpG methylation panel (E5‐α_3887, E5‐α_3941, E7_701, L2_4441, and L2_5128) as promising triage biomarkers for HPV16‐positive women, achieving high discriminatory performance (AUC = 0.919 in a validation cohort) for detecting cervical lesions. This genome‐wide capture sequencing identified novel HPV16 methylation markers that distinguish cervical lesions from normal tissue, supporting the feasibility of HPV methylation‐based triage for HPV‐positive women in cervical cancer screening.

The CRISPR‐Cas12a system has emerged as a promising tool for nucleic acid‐based diagnostics. However, its multi‐step workflow and limited sensitivity hinder its integration into point‐of‐care testing (POCT). Here, the ECOT system (Engineered Cas12a for One‐pot Test), a novel approach that combines protein engineering with one‐pot detection, offering high sensitivity, specificity, and rapid response is introduced. By introducing GCN4 epitope insertions into LtCas12a and LbCas12a variants, their cis ‐cleavage activity, promoting efficient accumulation of amplification products is reduced. Additionally, the inclusion of scFv‐Rad51 (single‐chain variable fragment‐Rad51) enhances Cas12a's trans ‐cleavage activity, amplifying signal intensity. The ECOT‐Lb system demonstrated superior sensitivity in detecting low‐copy HPV DNA samples, outperforming traditional qPCR in clinical tests. Achieving detection limits as low as 3 copies in under 30 min, the ECOT‐Lb system is well‐suited for home‐based self‐testing and widespread clinical diagnostics. This work provides a versatile and scalable protein engineering strategy that enhances the performance of CRISPR‐based diagnostic tools, offering a promising platform for rapid molecular detection in diverse applications.

CRISPR-Cas9 systems have revolutionized genome editing, but the off-target effects of Cas9 limit its use in clinical applications. Here, we systematically evaluate FrCas9, a variant from Faecalibaculum rodentium , for cell and gene therapy (CGT) applications and compare its performance to SpCas9 and OpenCRISPR-1. OpenCRISPR-1 is a CRISPR system synthesized de novo using large language models (LLMs) but has not yet undergone systematic characterization. Using AID-seq, Amplicon sequencing, and GUIDE-seq, we assessed the on-target activity and off-target profiles of these systems across multiple genomic loci. FrCas9 demonstrated higher on-target efficiency and substantially fewer off-target effects than SpCas9 and OpenCRISPR-1. Furthermore, TREX2 fusion with FrCas9 reduced large deletions and translocations, enhancing genomic stability. Through screening of 1903 sgRNAs targeting 21 CGT-relevant genes using sequential AID-seq, Amplicon sequencing, and GUIDE-seq analysis, we identified optimal sgRNAs for each gene. Our high-throughput screening platform highlights FrCas9, particularly in its TREX2-fused form, as a highly specific and efficient tool for precise therapeutic genome editing.

Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full-length PE effector size (6.3-kb) is beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical translation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag-PE system that splits PE effectors into GCN4-nCas9 and single-chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag-PEs with 1×GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag-PE strategy achieved editing levels comparable to canonical fused-PE (nCas9 and RT are linked together) and higher than other split-PE strategies (including sPE and MS2-PE) in both PE2 and PE3 forms with no increase in insertion and deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag-PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag-ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag-PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics.