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A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing.
The insertion of large genetic circuits and metabolic pathways into bacterial genomes is becoming increasingly common within the field of synthetic biology due to the improved robustness and stability that come with genome integration. CRISPR-associated transposases (CASTs) enable RNA-guided DNA insertion without introducing double-stranded breaks and have been shown to function across diverse bacterial species. Here, we present an improved tool called pSPIN-GG and supporting protocols for simplified CAST-based genome engineering. The pSPIN-GG system includes Golden Gate-compatible promoter, guide, and cargo modules for simple assembly, a green fluorescent protein dropout cassette for rapid verification of guide replacement, and a set of tested sites within the Escherichia coli BL21 chromosome to enable gene dosing of genetic cargoes. These refinements support accelerated library construction, reduce assembly and screening burden, and expand the accessibility of CAST systems for multiplexed bacterial genome engineering.
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Applying CRISPR Technologies for the Treatment of Human Herpesvirus Infections: A Scoping Review.
BACKGROUND: Human herpesviruses are double-stranded DNA viruses of which eight types have been identified at present. Herpesvirus infection comprises an active lytic phase and a lifelong latency phase with the possibility of reactivation. These infections are highly prevalent worldwide and can lead to a broad spectrum of clinical manifestations, ranging from mild symptoms to severe disease, particularly in immunocompromised individuals. Clustered regularly interspaced palindromic repeats (CRISPR)-based therapy is an interesting alternative to current antiviral drugs, which fail to cure latent infections and are increasingly challenged by viral resistance. OBJECTIVE: This scoping review aimed to summarize the current state of CRISPR-based antiviral strategies against herpesvirus infections, highlighting the underlying mechanisms, study design and outcomes, and challenges for clinical implementation. DESIGN: A literature search was conducted in the databases PubMed and Web of Science, using both a general and an individual approach for each herpesvirus. RESULTS: This scoping review identified five main mechanisms of CRISPR-based antiviral therapy against herpesvirus infections in vitro and/or in vivo. First, CRISPR systems can inhibit the active lytic replication cycle upon targeting viral lytic genes or host genes. Second, CRISPR technologies can remove latent viral genomes from infected cells by targeting viral genes essential for latency maintenance or destabilizing the viral genome. Third, reactivation of multiple latent herpesvirus infections can be inhibited by CRISPR-Cas-mediated editing of lytic viral genes, preventing a flare-up of clinical symptoms and reducing the risk of viral transmission. Fourth, CRISPR systems can purposefully induce viral reactivation to enhance recognition by the host immune system or improve the efficacy of existing antiviral therapies. Fifth, CRISPR technology can be applied to develop or enhance the efficiency of cellular immunotherapy. CONCLUSIONS: Multiple studies demonstrate the potential of CRISPR-based antiviral strategies to target herpesvirus infections through various mechanisms in vitro and in vivo. However, aspects regarding the delivery and biosafety of CRISPR systems, along with the time window for treatment, require further investigation before broad clinical implementation can be realized.
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Zebrafish and CRISPR-A synergistic approach to decipher and cure human diseases.
Rapidly emerging infectious and genetic diseases demand robust vertebrate models to investigate pathogenesis and accelerate therapeutic discovery. Zebrafish (Danio rerio) offer substantial translational value owing to their conserved physiology, optical transparency, rapid reproduction, and the presence of orthologs for approximately 70% of human genes and approximately 82% of disease-associated genes. The integration of CRISPR/Cas9 technology has transformed zebrafish research, enabling efficient generation of targeted knockouts, knockins, and high-throughput mutagenesis screens. This synergy supports mechanistic dissection and modeling of cardiovascular, oncologic, viral, and other genetic disorders. Despite these advantages, rigorous allele validation, consideration of paralog redundancy, maternal contribution, and off-target analysis remain essential to ensure translational accuracy. This review summarizes current applications, methodological advances, limitations, and best-practice recommendations for combining zebrafish models with genome editing to improve understanding and treatment of human diseases.
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Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
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CRISPR technology: The engine that drives cancer therapy.
CRISPR gene editing technology belongs to the third generation of gene editing technology. Since its discovery, it has attracted the attention of a large number of researchers. Investigators have published a series of academic articles and obtained breakthrough research results through in-depth research. In recent years, this technology has developed rapidly and been widely applied in many fields, especially in medicine. This review focuses on concepts of CRISPR gene editing technology, its application in cancer treatments, its existing limitations, and the new progress in recent years for detailed analysis and sharing.
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Single-Molecule Imaging of Telomerase RNA Reveals a Recruitment-Retention Model for Telomere Elongation.
Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.
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Deep Learning-Based Classification of CRISPR Loci Using Repeat Sequences.
With the widespread application of the CRISPR-Cas system in gene editing and related fields, along with the increasing availability of metagenomic data, the demand for detecting and classifying CRISPR-Cas systems in metagenomic data sets has grown significantly. Traditional classification methods for CRISPR-Cas systems primarily rely on identifying cas genes near CRISPR arrays. However, in cases where cas gene information is absent, such as in metagenomes or fragmented genome assemblies, traditional methods may fail. Here, we present a deep learning-based method, CRISPRclassify-CNN-Att, which classifies CRISPR loci solely based on repeat sequences. CRISPRclassify-CNN-Att utilizes convolutional neural networks (CNNs) and self-attention mechanisms to extract features from repeat sequences. It employs a stacking strategy to address the imbalance of samples across different subtypes and uses transfer learning to improve classification accuracy for subtypes with fewer samples. CRISPRclassify-CNN-Att demonstrates outstanding performance in classifying multiple subtypes, particularly those with larger sample sizes. Although CRISPR loci classification traditionally depends on cas genes, CRISPRclassify-CNN-Att offers a novel approach that serves as a significant complement to cas-based methods, enabling the classification of orphan or distant CRISPR loci. The proposed tool is freely accessible via https://github.com/Xingyu-Liao/CRISPRclassify-CNN-Att.
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Applications of CRISPR/Cas9 in the research of malignant musculoskeletal tumors.
BACKGROUND: Malignant tumors of the musculoskeletal system, especially osteosarcoma, Ewing sarcoma and rhabdomyosarcoma, pose a major threat to the lives and health of adolescents and children. Current treatments for musculoskeletal tumors mainly include surgery, chemotherapy, and radiotherapy. The problems of chemotherapy resistance, poor long-term outcome of radiotherapy, and the inherent toxicity and side effects of chemical drugs make it extremely urgent to seek new treatment strategies. MAIN TEXT: As a potent gene editing tool, the rapid development of CRISPR/Cas9 technology in recent years has prompted scientists to apply it to the study of musculoskeletal tumors. This review summarizes the application of CRISPR/Cas9 technology for the treatment of malignant musculoskeletal tumors, focusing on its essential role in the field of basic research. CONCLUSION: CRISPR, has demonstrated strong efficacy in targeting tumor-related genes, and its future application in the clinical treatment of musculoskeletal tumors is promising.
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Methods and applications of in vivo CRISPR screening.
A fundamental goal in genetics is to understand the connection between genotype and phenotype in health and disease. Genetic screens in which dozens to thousands of genetic elements are perturbed in a pooled fashion offer the opportunity to generate large-scale, information-rich and unbiased genotype-phenotype maps. Although typically applied in reductionist in vitro settings, methods enabling pooled CRISPR-Cas perturbation screening in vivo are gaining attention as they have the potential to accelerate the discovery and annotation of gene function across cells, tissues, developmental stages, disease states and species. In this Review, we discuss essential criteria for understanding, designing and implementing in vivo screening experiments, with a focus on pooled CRISPR-based screens in mice. We also highlight how the resulting datasets, combined with advances in multi-omics and artificial intelligence, will accelerate progress and enable fundamental discoveries across basic and translational sciences.
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Optimization of genome editing by CRISPR ribonucleoprotein for high efficiency of germline transmission of Sox9 in zebrafish.
Primordial germ cells (PGCs) are the first germline stem cells to emerge during early embryonic development and are essential for the propagation and survival of species. Genome editing creates mutagenesis possibilities in vivo, but the generation of precise mutations in PGCs is still challenging. Here, we report an optimized approach for highly efficient genome editing via introducing biallelic variations in early embryos in zebrafish. We adopted an extended, GC-rich, and chemically modified sgRNA along with microinjection of the CRISPR ribonucleoprotein (RNP) complex into the yolk sac at the 1-cell stage. We found that genome editing of Sox9a generated a high proportion of heterozygotes with edited alleles in the F1 generation, indicating biallelic editing. Deep sequencing and mapping the edited cells from early embryos to future tissues revealed that the edited founder has a dominantly edited allele, sox9a M1, accounting for over 99 % of alleles in the testis. Specifically, all offspring of the founder inherited the edited allele, suggesting nearly complete editing of the alleles in early germline cells. Overall, the optimization delineates biallelic editing of sox9a in early embryos and transmission of edited alleles to offspring, thus presenting a method to create a desired genetic mutation line of Sox9a avoiding lengthy traditional crossbreeding.
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