CRISPR-associated Protein Research Articles

Current developments of gene therapy in human diseases.

Gene therapy has witnessed substantial advancements in recent years, becoming a constructive tactic for treating various human diseases. This review presents a comprehensive overview of these developments, with a focus on their diverse applications in different disease contexts. It explores the evolution of gene delivery systems, encompassing viral (like adeno-associated virus; AAV) and nonviral approaches, and evaluates their inherent strengths and limitations. Moreover, the review delves into the progress made in targeting specific tissues and cell types, spanning the eye, liver, muscles, and central nervous system, among others, using these gene technologies. This targeted approach is crucial in addressing a broad spectrum of genetic disorders, such as inherited lysosomal storage diseases, neurodegenerative disorders, and cardiovascular diseases. Recent clinical trials and successful outcomes in gene therapy, particularly those involving AAV and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins, are highlighted, illuminating the transformative potentials of this approach in disease treatment. The review summarizes the current status of gene therapy, its prospects, and its capacity to significantly ameliorate patient outcomes and quality of life. By offering comprehensive analysis, this review provides invaluable insights for researchers, clinicians, and stakeholders, enriching the ongoing discourse on the trajectory of disease treatment.

Efficient CRISPR/Cas9-mediated ebony gene editing in the greater wax moth Galleria mellonella.

The greater wax moth, Galleria mellonella (Lepidoptera, Pyralidae), is a major bee pest that inflicts considerable harm on beehives, leading to economic losses. It also serves as a valuable resource insect and a model organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system plays a crucial role in improving economic insect breeding and developing efficient agricultural pest management systems in Lepidoptera. However, the CRISPR/Cas9 protocols have not been developed for G. mellonella. Here, the Gmebony knockout (KO) strain was established using the CRISPR/Cas9 genome editing system. We obtained Gmebony KO strain in the G4 generation, which took approximately 10 months. When compared with wild-type, the head, notum, and the terminal abdominal surface of 1st to 4th instar larvae in the KO strain changed from yellow to brown, and these regions of the KO strain gradually transformed into a black color from the 5th instar larvae, and the body color of the adult moth in the KO strain changed to black. The developmental period of the early larval and the following larval instars extended. The embryonic hatchability of the Gmebony KO strain was significantly decreased. The pupal body weight of the Gmebony KO strain was not affected. The feasibility of the CRISPR/Cas9 methodology was validated by single-target editing of Gmebony. Our findings provide the first evidence that the ebony gene can serve as a pigmentation reference gene for genetic modifications of G. mellonella. Meanwhile, it can be utilized in the development of genome editing control strategies and for gene function analyses in G. mellonella.

The expression of PD-L1 on tumor-derived exosomes enhances infiltration and anti-tumor activity of αCD3 × αPD-L1 bispecific antibody-armed T cells

Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.Graphical abstract

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can’t acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Therapeutic Gene Editing in Dyslipidemias.

Dyslipidemia, characterized by abnormal lipid levels in the blood, significantly escalates the risk of atherosclerotic cardiovascular disease and requires effective treatment strategies. While existing therapies can be effective, long-term adherence is often challenging. There has been an interest in developing enduring and more efficient solutions. In this context, gene editing, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, emerges as a groundbreaking approach, offering potential long-term control of dyslipidemia by directly modifying gene expression. This review delves into the mechanistic insights of various gene-editing tools. We comprehensively analyze various pre-clinical and clinical studies, evaluating the safety, efficacy, and therapeutic implications of gene editing in dyslipidemia management. Key genetic targets, such as low-density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9), angiopoietin-like protein 3 (ANGPTL3), apolipoprotein C3 (APOC3), and lipoprotein (a) (Lp(a)), known for their pivotal roles in lipid metabolism, are scrutinized. The paper highlights the promising outcomes of gene editing in achieving sustained lipid homeostasis, discusses the challenges and ethical considerations in genome editing, and envisions the future of gene therapy in revolutionizing dyslipidemia treatment and cardiovascular risk reduction.

Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the β654-Thalassemia Mouse Model.

β654-thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β654 mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β654-thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β654-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced β-globin mRNA. Pathological changes were improved compared with the nonedited β654 mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β654 allele. The therapeutic effects of this gene-editing strategy showed that the typical β-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a β654 mouse model.

CRISPR technology in human diseases.

Gene editing is a growing gene engineering technique that allows accurate editing of a broad spectrum of gene-regulated diseases to achieve curative treatment and also has the potential to be used as an adjunct to the conventional treatment of diseases. Gene editing technology, mainly based on clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein systems, which is capable of generating genetic modifications in somatic cells, provides a promising new strategy for gene therapy for a wide range of human diseases. Currently, gene editing technology shows great application prospects in a variety of human diseases, not only in therapeutic potential but also in the construction of animal models of human diseases. This paper describes the application of gene editing technology in hematological diseases, solid tumors, immune disorders, ophthalmological diseases, and metabolic diseases; focuses on the therapeutic strategies of gene editing technology in sickle cell disease; provides an overview of the role of gene editing technology in the construction of animal models of human diseases; and discusses the limitations of gene editing technology in the treatment of diseases, which is intended to provide an important reference for the applications of gene editing technology in the human disease.

NAD+ metabolism enzyme NNMT in cancer-associated fibroblasts drives tumor progression and resistance to immunotherapy by modulating macrophages in urothelial bladder cancer

BackgroundThis study comprehensively investigates the association between the expression of nicotinamide N-methyltransferase (NNMT) and clinical outcomes of urothelial bladder cancer (UBC), as well as the molecular mechanisms by which NNMT...

Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson's disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.

Targeted eradication of EBV-positive cancer cells by CRISPR/dCas9-mediated EBV reactivation in combination with ganciclovir.

This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.

Using CRISPR-Cas9 to create knockout mutants in insects

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) is a potent genome editing tool that has rapidly revolutionized research in natural science disciplines. The CRISPR/Cas9 system surpasses traditional zinc finger nucleases and transcription activator-like effector nucleases because of its simplicity in design, cost-effectiveness, and labor intensity. The CRISPR/Cas9-mediated genome editing system has been used to generate precise and targeted mutations in many different organisms, including insects. However, there is still a lack of a comprehensive review that systematically summarizes the successful utilization of the CRISPR/Cas9 system in generating knockout (KO) mutations in insects (both model and non-model). Thus, this review provides a comprehensive analysis of the current advancements in CRISPR/Cas9-based KO mutants in insects, including both model and non-model species. We also discussed the limitations of the CRISPR/Cas9 system. Finally, we discuss the biosafety concerns associated with the release of CRISPR/Cas9-edited insects into the environment and draw inroads and insights into the future application of the CRISPR/Cas9 system in insects.

A dynamic subpopulation of CRISPR-Cas overexpressers allows Streptococcus pyogenes to rapidly respond to phage.

Many CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems, which provide bacteria with adaptive immunity against phages, are transcriptionally repressed in their native hosts. How CRISPR-Cas expression is induced as needed, for example, during a bacteriophage infection, remains poorly understood. In Streptococcus pyogenes, a non-canonical guide RNA tracr-L directs Cas9 to autorepress its own promoter. Here we describe a dynamic subpopulation of cells harbouring single mutations that disrupt Cas9 binding and cause CRISPR-Cas overexpression. Cas9 actively expands this population by elevating mutation rates at the tracr-L target site. Overexpressers show higher rates of memory formation, stronger potency of old memories and a larger memory storage capacity relative to wild-type cells, which are surprisingly vulnerable to phage infection. However, in the absence of phage, CRISPR-Cas overexpression reduces fitness. We propose that CRISPR-Cas overexpressers are critical players in phage defence, enabling bacterial populations to mount rapid transcriptional responses to phage without requiring transient changes in any one cell.

Applications of CRISPR in Parasitology.

Clustered Regions of Interspersed Palindromic Repeat (CRISPR)-based techniques have been utilized in various research areas, including agriculture, biotechnology, and medicine. With the use of a short sequence guide RNA and CRISPR-associated (Cas) protein, this technique allows for robust, site-specific manipulation of the genome, aiding researchers in making important biomedical discoveries and scientific advancements. In this review, we explored the applications of CRISPR/Cas systems in the field of parasitology for the identification and validation of novel functional genes, diagnosis of parasitic infections, reduction of parasite virulence, and the disruption of disease transmission. We also discussed how CRISPR can be used for the development of therapeutics, vaccines, and drug discovery. Furthermore, the challenges and future perspectives of this technology are also highlighted.

Pathogen dynamics and discovery of novel viruses and enzymes by deep nucleic acid sequencing of wastewater

Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Exploiting CRISPR/Cas system in controlling antibiotic resistance of bacteria

The emergence and spread of antimicrobial resistance is a threat to human health, therefore there is a need to explore new strategies to combat antibiotic-resistant bacteria. Antimicrobial resistance genes are mainly transmitted through horizontal gene transfer (HGT) mediated by transposable elements, phages, and plasmids, which leads to the emergence of multidrug-resistant bacteria. Clustered Regularly interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) protect archaebacteria and eubacteria from potentially harmful foreign DNA such as plasmids, transposable elements, and phages. Several studies have introduced the CRISPR/Cas system as an innovative and effective strategy to trigger antibiotic resistance and virulent genes in bacteria. Therefore, this study aimed to introduce the CRISPR-Cas system in controlling antibiotic resistance of bacteria. We hope exploiting CRISPR/Cas could provide a bright future for the treatment of disastrous infections with resistant and virulent organisms.

Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

Application of genome editing in plant reproductive biology: recent advances and challenges.

This comprehensive review underscores the application of genome editing in plant reproductive biology, including recent advances and challenges associated with it. Genome editing (GE) is a powerful technology that has the potential to accelerate crop improvement by enabling efficient, precise, and rapid engineering of plant genomes. Over the last decade, this technology has rapidly evolved from the use of meganucleases (homing endonucleases), zinc-finger nucleases, transcription activator-like effector nucleases to the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas), which has emerged as a popular GE tool in recent times and has been extensively used in several organisms, including plants. GE has been successfully employed in several crops to improve plant reproductive traits. Improving crop reproductive traits is essential for crop yields and securing the world's food supplies. In this review, we discuss the application of GE in various aspects of plant reproductive biology, including its potential application in haploid induction, apomixis, parthenocarpy, development of male sterile lines, and the regulation of self-incompatibility. We also discuss current challenges and future prospects of this technology for crop improvement, focusing on plant reproduction.

Targeted protein editing technique in living mammalian cells by peptide-fused PNGase

Various precise gene editing techniques at the DNA/RNA level, driven by clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, have gained significant prominence. Yet, research on targeted protein editing techniques remains limited. Only a few attempts have been made, including the use of specific proteases and de-O-glycosylating enzymes as editing enzymes. Here, we propose direct editing of N-glycosylated proteins using de-N-glycosylating enzymes to modify N-glycosylation and simultaneously alter the relevant asparagine residue to aspartate in living cells. Selective protein deglycosylation editors were developed by fusing high-affinity protein-targeting peptides with active peptide:N-glycanases (PNGases). Three crucial cell membrane proteins, PD-1, PD-L1, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein, were chosen to be tested as a proof of concept. N-linked glycans were removed, and the relevant sites were converted from Asn to Asp in living mammalian cells, destabilizing target proteins and accelerating their degradation. Further investigation focused on SARS-CoV-2 spike protein deglycosylation editing. The collaboration of LCB1-PNGase F (PNGF) effectively reduced syncytia formation, inhibited pseudovirus packaging, and significantly hindered virus entry into host cells, which provides insights for coronavirus disease 2019 (COVID-19) treatment. This tool enables editing protein sequences post-de-N-glycosylation in living human cells, shedding light on protein N-glycosylation functions, and Asn to Asp editing in organisms. It also offers the potential for developing protein degradation technologies.

Investigating a role for PUB17 and PUB16 in the self-incompatibility signaling pathway in transgenic Arabidopsis thaliana.

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen S cysteine-rich/S-locus protein 11 (SCR/SP11) ligands and the stigma S receptor kinases (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARM-repeat containing 1 (ARC1), is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A.thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between A.thaliana accessions, and ARC1 is not always needed to produce a strong SI response. For the A.thaliana C24 accession, only transforming with Arabidopsis lyrata SCR and SRK confers a strong SI trait (SI-C24), and so here, we investigated if ARC1-related PUBs were involved in the SI pathway in the transgenic A.thaliana SI-C24 line. Two close ARC1 homologs, PUB17 and PUB16, were selected, and (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into the transgenic A.thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences in SI responses to implicate PUB17 and PUB16 functioning in the transgenic A.thaliana SI-C24 stigma to reject SI pollen.

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.

Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.