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.

Somatic evolution leads to clonal heterogeneity, which fuels cancer progression and therapy resistance. To decipher the consequences of clonal heterogeneity, we require a method that deconvolutes complex clonal architectures and their downstream transcriptional states. We developed Genotyping of Transcriptomes for multiple targets and sample types (GoT-Multi), a high-throughput, formalin-fixed paraffin-embedded (FFPE) tissue-compatible single-cell multi-omics for co-detection of multiple somatic genotypes and whole transcriptomes. We developed an ensemble-based machine learning pipeline to optimize genotyping. We applied GoT-Multi to frozen or FFPE samples of Richter transformation, a progression of chronic lymphocytic leukemia to therapy-resistant large B cell lymphoma. GoT-Multi detected heterogeneous cancer cell states with genotypic data of 27 mutations, enabling clonal architecture reconstruction linked with their transcriptional programs. Distinct subclonal genotypes, including therapy-resistant mutations, converged on an inflammatory state. Other subclones displayed enhanced proliferation and/or MYC program. Thus, GoT-Multi revealed that distinct genotypic identities may converge on similar transcriptional states to mediate therapy resistance.

Regulation of transcription initiation is the ground level of modulating gene expression during plant development. This process relies on interactions between transcription factors and cis-regulatory elements (CREs), which become promising targets for crop bioengineering. To annotate CREs in the barley genome and understand mechanisms of distal regulation, we profiled several epigenetic features across three stages of barley embryo and leaves and performed HiChIP to identify activating and repressive genomic interactions. Using machine learning, we integrated the data into seven chromatin states, predicting ∼77,000 CRE candidates, collectively representing 1.43% of the barley genome. Identified genomic interactions, often spanning multiple genes, linked thousands of predicted CREs with their putative targets and revealed notably frequent promoter-promoter contacts. Using the LEA gene family as an example, we discuss possible roles of these interactions in transcription regulation. On the Vrn3 gene, we demonstrate the potential of our datasets to predict CREs for other developmental stages.

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.

All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Here, we show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the turnover of subterminal repetitive DNA. We characterized the fusion site at the single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. One of these SDs shares homology to a hypomethylated SD spacer sequence present in the subterminal heterochromatin of Pan but is completely absent subtelomerically in both humans and orangutans. CRISPR-Cas9-mediated depletion of the fusion site in human neural progenitor cells alters the expression of genes, indicating a potential regulatory consequence to this human-specific karyotypic change. Overall, this study offers insights into how complex regions subject to ILS may contribute to speciation.

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.