Editing Tools Research Articles

CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.

Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.

Prime editing: A gene precision editing tool from inception to present.

Genetic mutations significantly contribute to the onset of diseases, with over half of the cases caused by single-nucleotide mutations. Advances in gene editing technologies have enabled precise editing and correction of mutated genes, offering effective treatment methods for genetic disorders. CRISPR/Cas9, despite its power, poses risks of inducing gene mutations due to DNA double-strand breaks (DSB). The advent of base editing (BE) and prime editing (PE) has mitigated these risks by eliminating the hazards associated with DNA DSBs, allowing for more precise gene editing. This breakthrough lays a solid foundation for the clinical application of gene editing technologies. This review discusses the principles, development, and applications of PE gene editing technology in various genetic mutation-induced diseases.

Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells.

The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.

Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a model organism for unicellular green microalgae, is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including (i) unsatisfactory genome editing (GE) efficiency and (ii) uncontrolled DNA random insertion of antibiotic resistance markers. Thus, obtaining efficient gene targeting without using marker genes is instrumental in developing a pipeline for efficient engineering of strains for biotechnological applications. We developed an efficient DNA-free gene disruption strategy, relying on phenotypical identification of mutants, to (i) precisely determine its efficiency compared to marker-relying approaches and (ii) establish a new DNA-free editing tool. This study found that classical CRISPR Cas9-based GE for gene disruption in Chlamydomonas reinhardtii is mainly limited by DNA integration. With respect to previous results achieved on synchronized cell populations, we succeeded in increasing the GE efficiency of single gene targeting by about 200 times and up to 270 times by applying phosphate starvation. Moreover, we determined the efficiency of multiplex simultaneous gene disruption by using an additional gene target whose knock-out did not lead to a visible phenotype, achieving a co-targeting efficiency of 22%. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families. Furthermore, the approach provides new perspectives on how GE can be applied to (non-) model microalgae species, targeting groups of candidate genes of high interest for basic research and biotechnological applications.

Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles

Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

(Dis)placed Contributions: Uncovering Hidden Hurdles to Collaborative Writing Involving Non-Native Speakers, Native Speakers, and AI-Powered Editing Tools

Content creation today often takes place via collaborative writing. A longstanding interest of CSCW research lies in understanding and promoting the coordination between co-writers. However, little attention has been paid to individuals who write in their non-native language and to co-writer groups involving them. We present a mixed-method study that fills the above gap. Our participants included 32 co-writer groups, each consisting of one native speaker (NS) of English and one non-native speaker (NNS) with limited proficiency. They performed collaborative writing adopting two different workflows: half of the groups began with NNSs taking the first editing turn and half had NNSs act after NSs. Our data revealed a 'late-mover disadvantage' exclusively experienced by NNSs: an NNS's ideational contributions to the joint document were suppressed when their editing turn was placed after an NS's turn, as opposed to ahead of it. Surprisingly, editing help provided by AI-powered tools did not exempt NNSs from being disadvantaged. Instead, it triggered NSs' overestimation of NNSs' English proficiency and agency displayed in the writing, introducing unintended tensions into the collaboration. These findings shed light on the fair assessment and effective promotion of a co-writer's contributions in language diverse settings. In particular, they underscore the necessity of disentangling contributions made to the ideational, expressional, and lexical aspects of the joint writing.

Gene Targeting via CRISPR/Cas9-Mediated DNA Double-Strand Break Induction in Rice.

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination. However, due to its low frequency, it has proved difficult to establish a universal GT system. The availability of the CRISPR/Cas9 system for genome editing in plants has opened up possibilities to apply GT successfully to some plant species. Here, we provide protocols for CRISPR/Cas9-mediated DNA double-strand break (DSB)-induced GT in rice.

Myh9b is a critical non-muscle myosin II encoding gene that interacts with myh9a and myh10 during zebrafish development in both compensatory and redundant pathways.

Non-muscle myosin (NMII) motor proteins have diverse developmental functions due to their roles in cell shape changes, cell migration, and cell adhesion. Zebrafish are an ideal vertebrate model system to study the NMII encoding myh genes and proteins due to high sequence homology, established gene editing tools, and rapid ex utero development. In humans, mutations in the NMII encoding MYH genes can lead to abnormal developmental processes and disease. This study utilized zebrafish myh9a, myh9b, and myh10 null mutants to examine potential genetic interactions and roles for each gene in development. It was determined that the myh9b gene is the most critical NMII encoding gene, as myh9b mutants develop pericardial edema and have a partially penetrant lethal phenotype, which was not observed in the other myh mutants. This study also established that genetic interactions occur between the zebrafish myh9a, myh9b, and myh10 genes where myh9b is required for the expression of both myh9a and myh10, and myh10 is required for the expression of myh9b. Additionally, protein analyses suggested that enhanced NMII protein stability in some mutant backgrounds may play a role in compensation. Finally, double mutant studies revealed different and more severe phenotypes at earlier timepoints than single mutants, suggesting roles for tissue specific genetic redundancy, and in some genotypes, haploinsufficiency. These mutants are the first in vivo models allowing for the study of complete loss of the NMIIA and NMIIB proteins, establishing them as valuable tools to elucidate the role of NMII encoding myh genes in development and disease.

Specifically Editing Cancer Sialoglycans for Enhanced In Vivo Immunotherapy through Aptamer‐Enzyme Chimeras

AbstractImmune checkpoint blockade (ICB) therapies have demonstrated remarkable clinical success in treating cancer. However, their objective response rate remains suboptimal because current therapies rely on limited immune checkpoints that fail to cover the multiple immune evasion pathways of cancer. To explore potential ICB strategies, we propose a glycoimmune checkpoint elimination (glycoICE) therapy based on targeted editing of sialoglycans on the tumor cell surface using an aptamer‐enzyme chimera (ApEC). The ApEC can be readily generated via a one‐step bioorthogonal procedure, allowing for large‐scale and uniform production. It specifically targets and desialylates cancer cells, disrupting the sialoglycan‐Siglec axis to activate immune cells and enhance immunotherapy efficacy, while its high tumor selectivity minimizes side effects from indiscriminate desialylation of normal tissues. Furthermore, the ApEC has the potential to be a versatile platform for specific editing of sialoglycans in different tumor models by adjusting the aptamer sequences to target specific protein markers. This research not only introduces a novel molecular tool for the effective editing of sialoglycans in complex environments, but also provides valuable insights for advancing DNA‐based drugs towards in vivo and clinical applications.

An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava.

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Mini and enhanced CRISPR activators for cancer therapies

IntroductionThe RNA-guided nuclease Cas9 can be used as a programmable transcription activator, but there is still room for improvement in its effectiveness in eukaryotes, and its potential in cancer genetic therapy has been poorly investigated. ObjectivesWe aim to construct optimized CRISPRa tools and detect their potential role in cancer therapy by screening 9aa-TAD. MethodsWe selected a range of transcriptional coactivators for programmable activation and analyzed their effects on the expression of multiple endogenous genes using Flow cytometry and qRT-PCR. In order to improve the activation capacity of the CRISPRa tool, we fused the coactivators with the efficient dCas9-VPR system to construct a new activation system. Utilize RNA-seq to assess the activation specificity of genome-wide. To evaluate the value of the newly constructed activation system in cancer gene therapy, we activated the expression of the tumor suppressor genes PER2 and ZNF382, and performed changes in cancer cell proliferation qRT-PCR and clonal formation analysis. ResultsIn this study, we screened the NHR module from C. elegans, which demonstrated a high transcription activation capacity with a compact size compared to VP64. We successfully demonstrated its efficiency in activating endogenous genes in mammalian cells. Furthermore, we developed an enhanced fused variant called NHR-VP64-p65-Rta (NVPR), which showed even higher efficiency compared to the previously established VPR module, making it an effective CRISPRa tool. The dCas9-NVPR complex also exhibited high specificity on a genome-wide scale. Finally, we utilized the dCas9-NVPR tool to restore the expression of tumor suppressor genes PER2 and ZNF382, effectively inhibiting the malignant phenotype of cancer cells. ConclusionWe have successfully developed and demonstrated a breakthrough CRISPRa tool with promising implications for cancer genetic therapy. This innovation expands the range of available gene editing tools and further validates the immense potential of CRISPR-based approaches in precision medicine.

Scoping review of countries’ practices missed by health emergency preparedness and response quantitative tools: COVID-19 and Ebola response experiences

IntroductionAfter the COVID-19 pandemic and previous disease outbreaks, it is critical to continuously examine the preparedness measurement tools and level of country preparedness for future health emergencies. Existing preparedness quantitative...

Recent Advances in Genetic Engineering Strategies of Sinorhizobium meliloti.

Sinorhizobium meliloti is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. S. meliloti has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B12. Advances in gene editing tools play a vital role in the development of S. meliloti strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in S. meliloti. This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in S. meliloti are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of S. meliloti, and serve as a reference for the utilization of genome editing tools for other Rhizobium species.

Corneal Endothelial Microscopy: Does a Manual Recognition of the Endothelial Cells Help the Morphometric Analysis Compared to a Fully Automatic Approach?

This study investigated whether manual integration in the recognition of the endothelial cells produces different outcomes of morphometric parameters compared to a fully automatic approach. Eight hundred and ninety endothelial images, originally acquired by the Perseus Specular Microscope (CSO, Florence, Italy), from seven positions of right and left corneas were selected from the database of the Research Centre in Optics and Optometry at the University of Milano-Bicocca. For each image selected, two procedures of cell identification were performed by the Perseus: an automatic identification and a manual-integrated procedure to add potential additional cells with the available editing tool. At the end of both procedures, the endothelial cell density (ECD), coefficient of variation (CV), and hexagonality (HEX) of the mosaic were calculated. The HEX in the two procedures was significantly different for all comparisons (p < 0.001), but clinically negligible. No significant differences were found for the CV and ECD in the images of both eyes irrespective of the corneal position of acquisition (except for ECD in three corneal portions, p < 0.05). To conclude, it is possible to recognise a significantly higher number of cells using the manual-integrated procedure than it is using the fully automatic one, but this does not change the morphological parameters achieved.

CRISPR System and Its Applications in Medicine and Stem Cell Engineering

Context: The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is a groundbreaking gene-editing tool that shows great promise for modifying genomes. Derived from prokaryotic adaptive immune defense mechanisms, this technique has been used in research on human diseases, demonstrating remarkable therapeutic potential. Through CRISPR, specific genetic mutations in patients can be corrected during gene therapy, offering a solution for treating diseases that were previously untreatable using conventional methods. This review explores the recent progress and future prospects of the CRISPR system, focusing on its applications in medicine and stem cell engineering. Special emphasis is placed on medical applications, the latest target design or analysis tools for genome editing, advancements in stem cell engineering, and associated innovations and challenges. Evidence Acquisition: This study reviewed articles indexed in ISI, SID, PubMed, and PubMed Central from 2007 to 2024. Results: Cas9, a key protein in CRISPR gene editing, is an endonuclease capable of targeting and cutting specific DNA sequences, guided by short RNA sequences. The gene editing process involves homology-directed repair (HDR), non-homologous end joining (NHEJ), and base editing pathways. Base editing, which modifies the epigenome without inducing DNA breaks, is gaining increasing attention. However, CRISPR still faces technical challenges, and the development of more efficient "super" CRISPR technology will likely require time. This article reviews the effectiveness, limitations, and applications of the CRISPR system. Conclusions: CRISPR/Cas9 tools allow for the creation of precise models, leading to more effective treatment options for patients.

CRISPR-Cas9: A Cutting-edge Genome-editing Technology with Many Potential Applications

Unregulated human activities are responsible for climate change which is a major factor for the emergence of new infectious (bacterial, viral, fungus, and parasitic) and non-infectious diseases (genetic disorders, cancers, etc.). To tackle this situation, we must have better therapeutics, diagnostics, and vaccines for the treatment and prevention of emerging diseases in humans and animals. To address the aforementioned issues, CRISPR-Cas9, a revolutionary genome editing tool, can help us develop better therapeutics, diagnostics, and vaccines in a short time. In addition, it can be used to achieve food security by improving productivity, adaptability, and resilience traits in plants and animals. In nature, most bacteria and archaea have CRISPR as a part of their adaptive immune system that helps bacteria defend themselves from phages, viruses, and other foreign genetic elements. CRISPR-Cas9 is a highly effective, simple, and accurate genome editing tool compared to previous genome editing tools such as meganucleases, Zinc finger nucleases (ZFNs), and transcription activators like effector nucleases (TALENs). This review article discusses the different components and working of the CRISPR-Cas9 system, and how CRISPR-Cas9 plays a significant role in the development of next-generation therapeutics, diagnostics, vaccine platforms, and improved crops and livestock species for food security.

Advances in CRISPR-Cas9 Technology for Tumor Therapy

Genome editing technology is an emerging toolbox that can specifically target genes. At present, the existing gene editing technologies include ZFN, TALEN, and CRISPR system editing technology with clusters of regularly spaced short palindromic repeats. However, ZFN and TALEN are no longer the preferred tools for gene editing due to some of their problems, and their complex protein design, high cost, and high difficulty are the main reasons that limit their use. The CRISPR system, on the other hand, is able to achieve specific binding by base complementary pairing of sgRNA to the target sequence. Traditional cancer treatment methods have limited efficacy and high recurrence rates, and the disease can be fundamentally treated by inactivating oncogenes or activating tumor suppressor genes through gene editing technology and then changing downstream signaling pathways for cancer treatment. With CRISPR-Cas9 technology, it is possible to have gene knockouts, insertions, and mutations. Here, we will investigate and explore the feasibility of the CRISPR system for tumor treatment, including the following three topics: oncogene knockout, augmentation of tumor suppressor gene expression, and enhancement of engineered T cell viability for effective tumor Through these methods, the CRISPR system is of great help and promising tumor therapy.

Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference

CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools.

CRISPR/Cas System in Human Genetic Diseases

Gene editing involves altering a particular sequence within an organism’s genome using gene-editing techniques. Efficiently and accurately insert, delete or replace genes to alter their genetic information and phenotypic characteristics. DNA nuclease-based gene editing technology has advanced rapidly, from the first-generation editing system ZFNs, the second-generation TALENs to the third-generation CRISPR/Cas9 system, the efficiency of gene editing has been continuously improved, the cost has been gradually reduced, and the application scope has been expanding. The new classification system categorizes CRISPR-Cas proteins into Class I and II as the two main classes. Class I encompasses several subtypes, including Type I, III, and IV, all form complexes through the coordinated function of multiple Cas proteins. While Class II includes Type II, Type V, and Type VI, which all rely on single large Cas protein to perform all response. By designing single guide RNA (sgRNA), CRISPR can be used to target any gene sequence intended to be edited, achieving the wanted therapeutic outcome in treatment of inherited genetic disease. Although potential for off-target leads to undesired genetic alterations even result in oncogenesis, CRISPR-Cas still works as powerful therapy and gene editing tool in all aspects. Future research requires us to solve off-target issue. This review systematically introduces CRISPR-Cas systems as promising therapeutic strategy towards various genetic disease, explaining molecular mechanism and classification of CRISPR and differences among them.

Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon.

CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (PDS) gene in melon. The constructed vectors were delivered to host plants using Agrobacterium-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a PDS editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.