Cell genomics最新文献

The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.

Genetic and epigenetic variation in enhancers is associated with disease susceptibility; however, linking enhancers to target genes and predicting enhancer dysfunction remain challenging. We mapped enhancer-promoter interactions in human pancreas using 3D chromatin assays across 28 donors and five cell types. Using a network approach, we parsed these interactions into enhancer-promoter tree models, enabling quantitative, genome-wide analysis of enhancer connectivity. A machine learning algorithm built on these trees estimated enhancer contributions to cell-type-specific gene expression. To test predictions, we perturbed enhancers in primary human pancreas cells with CRISPR interference and quantified effects at single-cell resolution using RNA fluorescence in situ hybridization (FISH) and high-throughput imaging. Tree models also annotated germline risk variants linked to pancreatic disorders, connecting them to candidate target genes. For pancreatic ductal adenocarcinoma risk, acinar regulatory elements showed greater variant enrichment, challenging the ductal cell-of-origin view. Together, these datasets and models provide a resource for studying pancreatic disease genetics.

Transcriptome-wide association studies (TWASs) are widely used to prioritize genes for diseases. Current methods test gene-disease associations at the bulk tissue or cell-type-specific pseudobulk level, which do not account for the heterogeneity within cell types. We present TWiST, a statistical method for TWAS at cell-state resolution using single-cell expression quantitative trait locus (eQTL) data. Our method uses pseudotime to represent cell states and models the effect of gene expression on the trait as a continuous pseudotemporal curve. Therefore, it allows flexible hypothesis testing of global, dynamic, and nonlinear associations. Through simulation studies and real data analysis, we demonstrated that TWiST leads to significantly improved power compared to pseudobulk methods. Application to the OneK1K study identified hundreds of genes with dynamic effects on autoimmune diseases along the trajectory of immune cell differentiation. TWiST presents great promise to understand disease genetics using single-cell studies.

Oocyte/embryo defects can result in oocyte maturation arrest, fertilization failure, embryonic arrest, and infertility as well as recurrent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) failures. However, the genetic determinants of human oocyte/embryo defects remain largely unknown, and the overall genetic diagnostic yield for such defects has not been evaluated. Here, we performed exome sequencing in 3,627 patients with oocyte/embryo defects. We identified a total of 479 positive cases carrying variants in 37 known genes, indicating a diagnostic yield of 13.2%. Case-control association studies combined with gene set enrichment analysis identified 123 novel candidate genes responsible for oocyte/embryo defects. These results provide a comprehensive genetic landscape of human oocyte/embryo defects and highlight the clinical significance of genetic counseling in infertile patients with oocyte/embryo defects. Our study will lay the foundation for transforming the traditional clinical practice for failed IVF/ICSI attempts into genetic-based precision and personalized treatment for these patients.

The human immunoglobulin heavy chain constant (IGHC) domain of antibodies (Abs) is responsible for effector functions critical to immunity. This domain is encoded by genes in the IGHC locus, where descriptions of genomic diversity remain incomplete. We utilized long-read sequencing to build an IGHC haplotype/variant catalog from 105 individuals of diverse ancestry. We discovered uncharacterized single-nucleotide variants (SNVs) and large structural variants (SVs; n = 7) representing new genes and alleles enriched for non-synonymous substitutions, highlighting potential functional effects. Of the 221 identified IGHC alleles, 192 were novel. SNV, SV, and gene allele/genotype frequencies revealed population differentiation, including (1) hundreds of SNVs in African and East Asian populations exceeding a fixation index (F_ST) of 0.3 and (2) an IGHG4 haplotype carrying coding variants uniquely enriched in Asian populations. Our results illuminate missing signatures of IGHC diversity and establish a new foundation for investigating IGHC germline variation in Ab function and disease.

To trace the history of human-cat interactions and the arrival of domestic cats (Felis catus) in East Asia, we analyzed 22 small felid bones excavated from 14 archaeological sites across China spanning 5,000 years. Genomic and radiocarbon evidence revealed that commensal leopard cats (Prionailurus bengalensis) appeared in anthropogenic environments at least 5,400 years ago and persisted until 150 CE. After a gap of several centuries, the earliest known domestic cat in China (c. 730 CE), reconstructed as a fully or partially white cat, was identified in Shaanxi during the Tang Dynasty. Genomic analysis combining 130 modern and ancient Eurasian cat specimens suggested an origin of Chinese domestic cats from the Levant and a likely merchant-mediated dispersal via the Silk Road. Commensal leopard cats and domestic cats once independently inhabited ancient human settlements in China but followed divergent sociocultural paths with only domestic cats becoming fully domesticated and globally introduced.

According to the current paradigm, human monogenic disorders underlying immunological phenotypes are due to rare (frequency <1%) as opposed to common (>1%) alleles. However, as reviewed here, an increasing number of studies have reported monogenic disorders of immunity, recessive or dominant, involving alleles that are currently common in specific small or large populations. Examples range from IFNAR1 and IFNAR2 null alleles in the Arctic and Pacific to PTCRA hypomorphic alleles in South Asia. This situation may be explained by a history of (1) population bottlenecks followed by expansion; (2) genetic drift before the advent of an environmental trigger; (3) slow purging, especially for recessive, mild, or incompletely penetrant conditions; and/or (4) balancing selection with a heterozygous advantage. In patients with suspected monogenic immunological conditions, a role for alleles common in the corresponding population should not be excluded. At odds with the prevailing view, common alleles may underlie monogenic disorders of immunity and should therefore be considered.

Tracking clonal evolution is critical to fully define the mechanisms of normal physiology and the disruptions of these processes in disease. In this issue of Cell Genomics, Pak and Saurty-Seerunghen et al. describe the development of Genotyping of Transcriptomes for multiple targets and sample types (GoT-Multi) and show how this new technology enables insights into cellular states that mediate clonal evolution in diseases, such as the Richter transformation of chronic lymphocytic leukemia, while also revealing convergence of cell states, even with distinct driver mutations.

The advent of long-read sequencing and telomere-to-telomere (T2T) assemblies has transformed studies of eukaryotic genomic variation. Pangenomes now leverage these advances to generate comprehensive catalogs of structural variants (SVs) and gene presence-absence polymorphisms across populations. Here, we review how pangenomes improve the identification, classification, and large-scale analysis of SVs and gene families, yielding insights into genome organization, functional gene evolution, and the architecture of phenotypic traits. We discuss mechanisms of SV formation, their uneven genomic distribution, and their roles in trait diversity. Examples from humans, plants, animals, and fungi highlight the importance of SVs in adaptation, domestication, and disease. We also consider the integration of pangenome graphs into genome-wide association studies, the challenges of applying T2T pangenomes at the population scale, and the need for new computational tools. Together, pangenomes represent a transformative framework for decoding genomic diversity and its consequences.

Tumor-specific antigens (TSAs) are crucial for activating T cells against cancer, but traditional discovery methods focusing on exonic mutations overlook non-canonical TSAs from non-coding regions. We employed an integrative proteogenomic strategy combining whole-genome and RNA sequencing with immunoprecipitation mass spectrometry to comprehensively explore TSA generation in colorectal cancer patients. Analysis of 10 paired tumor samples identified 96 mutated major histocompatibility complex class I-presented neo-epitopes, with 80.21% originating from non-coding regions. In hypermutated tumors with high mutational burden, neo-epitopes predominantly arose from intergenic and intronic areas, while in non-hypermutated tumors with low mutational burden, they mainly stemmed from coding variations and alternative splicing events. Functional validation in mouse models demonstrated that mutated non-canonical neo-epitopes effectively activated CD8⁺ T cells and significantly suppressed tumor growth. These findings underscore the importance of considering the entire genomic landscape in TSA discovery, suggesting new avenues for personalized immunotherapy.