CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Single-cell senescence identification reveals senescence heterogeneity, trajectory, and modulators.

Cellular senescence underlies many aging-related pathologies, but its heterogeneity poses challenges for studying and targeting senescent cells. We present here a machine learning program senescent cell identification (SenCID), which accurately identifies senescent cells in both bulk and single-cell transcriptome. Trained on 602 samples from 52 senescence transcriptome datasets spanning 30 cell types, SenCID identifies six major senescence identities (SIDs). Different SIDs exhibit different senescence baselines, stemness, gene functions, and responses to senolytics. SenCID enables the reconstruction of senescent trajectories under normal aging, chronic diseases, and COVID-19. Additionally, when applied to single-cell Perturb-seq data, SenCID helps reveal a hierarchy of senescence modulators. Overall, SenCID is an essential tool for precise single-cell analysis of cellular senescence, enabling targeted interventions against senescent cells.

Identification of the metaphyseal skeletal stem cell building trabecular bone.

Skeletal stem cells (SSCs) that are capable of self-renewal and multipotent differentiation contribute to bone development and homeostasis. Several populations of SSCs at different skeletal sites have been reported. Here, we identify a metaphyseal SSC (mpSSC) population whose transcriptional landscape is distinct from other bone mesenchymal stromal cells (BMSCs). These mpSSCs are marked by Sstr2 or Pdgfrb+Kitl-, located just underneath the growth plate, and exclusively derived from hypertrophic chondrocytes (HCs). These HC-derived mpSSCs have properties of self-renewal and multipotency in vitro and in vivo, producing most HC offspring postnatally. HC-specific deletion of Hgs, a component of the endosomal sorting complex required for transport, impairs the HC-to-mpSSC conversion and compromises trabecular bone formation. Thus, mpSSC is the major source of BMSCs and osteoblasts in bone marrow, supporting the postnatal trabecular bone formation.

Transformer for one stop interpretable cell type annotation.

Consistent annotation transfer from reference dataset to query dataset is fundamental to the development and reproducibility of single-cell research. Compared with traditional annotation methods, deep learning based methods are faster and more automated. A series of useful single cell analysis tools based on autoencoder architecture have been developed but these struggle to strike a balance between depth and interpretability. Here, we present TOSICA, a multi-head self-attention deep learning model based on Transformer that enables interpretable cell type annotation using biologically understandable entities, such as pathways or regulons. We show that TOSICA achieves fast and accurate one-stop annotation and batch-insensitive integration while providing biologically interpretable insights for understanding cellular behavior during development and disease progressions. We demonstrate TOSICA's advantages by applying it to scRNA-seq data of tumor-infiltrating immune cells, and CD14+ monocytes in COVID-19 to reveal rare cell types, heterogeneity and dynamic trajectories associated with disease progression and severity.

Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities.

Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine base editors (ABEs) do not introduce DNA double-strand breaks and thus have been proposed as alternatives to circumvent those problems, but their off-target effects still limit their applications. Here, we tested different combinations of off-target reduction methods to further diminish off-target effects of ABEs without compromising their on-target editing efficiencies. We subsequently chose the best editor, CE-8e-dV, which contains V106W substitution, R153 deletion, and Cas-embedding strategy, to establish a single-cell-derived human embryonic stem cell (hESC) line expressing tetracycline-inducible CE-8e-dV. By performing RNA and whole-genome sequencing, we demonstrated that the expression of CE-8e-dV did not produce nearly any DNA or RNA off-target effects in hESCs. Our results provide stringent proof of the safety of ABEs in PSCs and suggest that CE-8e-dV could be suitable for related therapeutic strategies, such as generation of engineered stem cells in vitro and gene therapy in vivo.

Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA.

The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.

Engineering of near-PAMless adenine base editor with enhanced editing activity and reduced off-target.

About 47% of pathogenic point mutations could be corrected by ABE-induced A·T-to-G·C conversions. However, the applications of ABEs are still hindered by undesired editing efficiency, limited editing scopes, and off-targeting effects. Here, we develop a new adenine base editor, by embedding TadA-8e monomer into SpRY-nCas9, named as CE-8e-SpRY, which exhibits higher activity at NRN than NYN PAMs favored by SpRY nuclease. CE-8e-SpRY could target nearly all genomic sites in principle and induces the highest targeting efficiency among tested SpRY-based ABEs. In addition, CE-8e-SpRY also shows reduced RNA and DNA off-targeting activities. With optimized sgRNAs, CE-8e-SpRY induces efficient or desired target editing at some disease-relevant loci where conventional ABEs were unable to induce precise and satisfied editing. Taken together, our CE-8e-SpRY could broaden the applicability of ABEs in correcting or introducing pathogenic point mutations.

3D chromatin structure changes during spermatogenesis and oogenesis.

Gametogenesis, including spermatogenesis and oogenesis, are unique differentiation processes involving extraordinarily complex and precise regulatory mechanisms that require the interactions of multiple cell types, hormones, paracrine factors, genes and epigenetic regulators, and extensive chromatin 3D structure re-organization. In recent years, the development of 3D genome technology represented by Hi-C, enabled mapping of the 3D re-organization of chromosomes during zygogenesis at an unprecedented resolution. The 3D remodeling is achieved by folding chromatin into loops, topologically associating domains (TADs), and compartments (A and B), which ultimately affect transcriptional activity. In this review, we summarize the research progresses and findings on chromatin 3D structure changes during spermatogenesis and oogenesis.

Neddylation is essential for ß-catenin degradation in Wnt signaling pathway.

ß-Catenin is a central component in the Wnt signaling pathway; its degradation has been tightly connected to ubiquitylation, but it is rarely examined by loss-of-function assays. Here we observe that endogenous ß-catenin is not stabilized upon ubiquitylation depletion by a ubiquitylation inhibitor, TAK-243. We demonstrate that N-terminal phosphorylated ß-catenin is quickly and strongly stabilized by a specific neddylation inhibitor, MLN4924, in all examined cell types, and that ß-catenin and TCF4 interaction is strongly enhanced by inhibition of neddylation but not ubiquitylation. We also confirm that the E3 ligase ß-TrCP2, but not ß-TrCP1, is associated with neddylation and destruction of ß-catenin. GSK3ß and adenomatous polyposis coli (APC) are not required for ß-catenin neddylation but essential for its subsequent degradation. Our findings not only clarify the process of ß-catenin modification and degradation in the Wnt signaling pathway but also highlight the importance of reassessing previously identified ubiquitylation substrates.

CABE-RY: A PAM-flexible dual-mutation base editor for reliable modeling of multi-nucleotide variants.

Multi-nucleotide variants (MNVs) represent an important type of genetic variation and have biological and clinical significance. To simulate MNVs, we designed four dual-mutation base editors combining hA3A(Y130F), TadA8e(V106W), and protospacer adjacent motif (PAM)-flexible SpRY and selected cytosine and adenine base editor-SpRY (CABE-RY), which had the best editing performance, for further study. Characterization and comparison showed that CABE-RY had a smaller DNA editing window and lower RNA off-target edits than the corresponding single base editors. Thus, we have established a versatile tool to efficiently simulate MNVs over the genome, which could be very useful for functional studies on MNVs in humans.

A Cas-embedding strategy for minimizing off-target effects of DNA base editors.

DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.

Individual variation of the SARS-CoV-2 receptor ACE2 gene expression and regulation.

The COVID-19 coronavirus is now spreading worldwide. Its pathogen, SARS-CoV-2, has been shown to use angiotensin-converting enzyme 2 (ACE2) as its host cell receptor, same as the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. Epidemiology studies found males although only slightly more likely to be infected than females account for the majority of the severely ill and fatality, which also bias for people older than 60 years or with metabolic and cardiovascular diseases. Here by analyzing GTEx and other public data in 30 tissues across thousands of individuals, we found a significantly higher level in Asian females, an age-dependent decrease in all ethnic groups, and a highly significant decrease in type II diabetic patients of ACE2 expression. Consistently, the most significant expression quantitative loci (eQTLs) contributing to high ACE2 expression are close to 100% in East Asians, >30% higher than other ethnic groups. A shockingly common enrichment of viral infection pathways was found among ACE2 anti-expressed genes, and multiple binding sites of virus infection related transcription factors and sex hormone receptors locate at ACE2 regulatory regions. Human and mice data analysis further revealed ACE2 expression is reduced in T2D patients and with inflammatory cytokine treatment and upregulated by estrogen and androgen (both decrease with age). Our findings revealed a negative correlation between ACE2 expression and COVID-19 fatality at both population and molecular levels. These results will be instrumental when designing potential prevention and treatment strategies for ACE2 binding coronaviruses in general.

Estimating the contribution of bacteriophage to the dissemination of antibiotic resistance genes in pig feces.

The transfer of antibiotic resistance genes (ARGs) in the environment is a threat to both human and animal health. However, the contribution of bacteriophages to the dissemination of resistance genes via transduction is rarely explored. In this study, we screened pig feces from three commercial farms in China for 32 clinically relevant ARG types to assess the presence of the ARG population in bacteria and bacteriophage and further to estimate the contribution of bacteriophages to the dissemination of antibiotic resistance. We found that bacteriophage DNA contained 35.5% of the target ARG types and sul1, blaTEM and ermB were found in 100% of the phage DNA samples. The most abundant genes in the bacterial population were ermB and fexA whereas ermB was the most abundant in bacteriophage. In contrast, floR was the least abundant ARG in both populations. Also, the ratio index of the abundance of ARGs in bacteriophage and bacteria was firstly used in this study as an estimator of bacteriophage ability to transmit ARGs. The ratio for qnrA was the greatest (about 10-1) and differed from the most abundant bacteriophage ARG ermB. In addition, fexA had the lowest ratio value (about 10-6) and not floR. These results illustrate that ARGs abundance and detection rates used alone probably be not suitable for comprehensively judging the contribution of bacteriophage to the dissemination of antibiotic resistance. A more suitable model is the application of three indices; occurrence rate, absolute abundance in bacteriophage and the ratio value as warning and monitoring tools for environmental ARG assessments in bacteriophages.