Targeted Gene Editing Research Articles

Genome-Wide Genetic Architecture for Common Scab (Streptomyces scabei L.) Resistance in Diploid Potatoes

Most cultivated potato (Solanum tuberosum) varieties are highly susceptible to common scab (Streptomyces scabei). The disease is widespread in all major potato production areas and leads to high economic losses and food waste. Varietal resistance is seen as the most viable and sustainable long-term management strategy. However, resistant potato varieties are scarce, and their genetic architecture and resistance mechanisms are poorly understood. Moreover, diploid potato relatives to commercial potatoes remain to be fully explored. In the current study, a panel of 384 ethyl methane sulfonate (EMS)-mutagenized diploid potato clones were evaluated for common scab coverage, severity, and incidence traits under field conditions, and genome-wide association studies (GWASs) were conducted to dissect the genetic architecture of their traits. Using the GAPIT-MLM and RTM-GWAS statistical models, and Mann–Whitney non-parametric U-tests, we show that 58 QTNs/QTLs distributed on all 12 potato chromosomes were associated with common scab resistance, 52 of which had significant allelic effects on the three traits. In total, 38 of the 52 favorable QTNs/QTLs were found to be pleiotropic on at least two of the traits, while 14 were unique to a single trait and were found distributed over 3 chromosomes. The identified QTNs/QTLs showed low to high effects, highlighting the quantitative and multigenic inheritance of common scab resistance. The QTLs/QTNs associated with the three common scab traits were found to be co-located in genomic regions carrying 79 candidate genes playing roles in plant defense, cell wall component biosynthesis and modification, plant–pathogen interactions, and hormone signaling. A total of 61 potato clones were found to be tolerant or resistant to common scab. Taken together, the data show that the studied germplasm panel, the identified QTNs/QTLs, and the candidate genes are prime genetic resources for breeders and biologists in breeding and targeted gene editing.

The Implication and Advancement of CRISPR Genome Editing in Microalgae and Cyanobacteria

This review paper provides an overview of recent achievements and future prospects of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-Cas9) genome editing in microalgae and cyanobacteria. The different types of CRISPR systems and Plasmid-based approaches, RNP-based Cas9 expression, transient and stable expression of Cas9 and sgRNA, and various other techniques for targeted gene editing have been reviewed. The paper also highlights the achievements of CRISPR-Cas9 genome editing in different cyanobacteria and algae species. Additionally, the challenges of off-target effects and potential solutions have been discussed. The paper concludes future prospects of CRISPR-Cas9 genome editing in microalgae and cyanobacteria, including gene stacking, markerless genome editing, and curing of episomes.

Optimization of Shoot Regeneration and Agrobacterium-mediated Transformation in Strawberry (Fragaria ×ananassa)

Efficient methods of plant transformation and tissue culture are essential for the genetic engineering and advanced molecular breeding of plants, but neither is well established in cultivated octoploid strawberry (Fragaria ×ananassa). In the present study, a method for shoot regeneration was established and optimized for two genetically different strawberry cultivars, Sweet Sensation® Florida 127 (FL127) and Florida Brilliance (FB). Runner segments at the tip, node, and petiole obtained from greenhouse-grown plants were used as explants for comparisons of shoot regeneration rate. ‘FL127’ showed the highest frequency of shoot regeneration in the optimized condition, whereas ‘FB’ showed the best response to a lower concentration of N6-benzyladenine (BA) (0.01 mg/L) in the same media type. The average conversion frequencies of somatic embryos into shoot regenerations from the runner tips (RTs) were 42.8% in ‘FL127’ and 56.9% in ‘FB’, respectively. Using these optimized tissue culture conditions, Agrobacterium-mediated CRISPR/Cas9 gene editing was conducted to examine the efficiency of transformation and target gene editing for the phytoene desaturase, FaPDS, in the cultivar, FL127. A total of 234 explants inoculated with Agrobacterium harboring Cas9-FaPDS resulted in an 80.3% callus induction efficiency, with 13.3% of regenerated plants exhibiting partial or complete albino phenotypes. Amplicon sequencing of edited progeny showed mutations (substitutions, insertions, and deletions) at the guide RNA (gRNA) target sites or flanking regions of all FaPDS homoeologous copies. Our results provide effective tissue culture and transformation methods for strawberry functional genomics research and gene editing guided cultivar improvement.

CRISPR Development and Application

Gene editing technology rapidly develop, addressing diseases that cannot be treated with conventional medical methods, nowadays, gene modification has become a hotspot of current research. A variety of methods utilizing DNA damage repair mechanisms to achieve targeted gene editing have gradually emerged. The CRISPR, which has been continuously optimized and improved since its development, has surpassed the previous two generations and become the third generation technology with more practical value., CRISPR technology compared to the previous two generations of technology have significant advancements in terms of application scope, specificity, and accuracy. CRISPR technology originates from bacteria themselves. As an acquired immune system of bacteria, it is used to identify intruding gene fragments and degrade them.The main content of this article includes the mechanism of CRISPR, functioning in bacteria and current popular classification methods for CRISPR systems. Focus on introducing, the delivery mode of CRISPR system in practical applications, like AAV, AdV or LV-based methods. And limitations of the current delivery mechanism and current development trends, that is to address the immunogenicity issues caused by viral vectors, researchers are continuously developing non-viral vectors. They have been made progress in many directions. It also introduces the current limitations of CRISPR/Cas9 itself. Especially addressing the issue of its high miss rate and the improvements made by scientists on this at present, such as developing high-fidelity variants of CRISPR/Cas9. In modern times, researchers have developed various artificial Cas9 variants that have similar functions to wild-type Cas9 and possess higher practical value.

Exosome-mediated delivery of CRISPR-Cas9: A revolutionary approach to cancer gene editing.

Several molecular strategies based on targeted gene delivery systems have been developed in recent years; however, the CRISPR-Cas9 technology introduced a new era of targeted gene editing, precisely modifying oncogenes, tumor suppressor genes, and other regulatory genes involved in carcinogenesis. However, efficiently and safely delivering CRISPR-Cas9 to cancer cells across the cell membrane and the nucleus is still challenging. Using viral vectors and nanoparticles presents issues of immunogenicity, off-target effects, and low targeting affinity. Naturally, extracellular vesicles called exosomes have garnered the most attention as delivery vehicles in oncology-related CRISPR-Cas9 calls due to their biocompatibility, loading capacity, and inherent targeting features. The following review discusses the current progress in using exosomes to deliver CRISPR-Cas9 components, the approaches to load the CRISPR components into exosomes, and the modification of exosomes to increase stability and tumor-targeted delivery. We discuss the latest strategies in targeting recently accomplished in the exosome field, including modifying the surface of exosomes to enhance their internalization by cancer cells, as well as the measures taken to overcome the impacts of TME on delivery efficiency. Focusing on in vitro and in vivo experimentation, this review shows that exosome-mediated CRISPR-Cas9 can potentially treat cancer types, including pancreatic, lymphoma, and leukemia, for given gene targets. This paper compares exosome-mediated delivery and conventional vectors regarding safety, immune response, and targeting ability. Last but not least, we present the major drawbacks and potential development of the seemingly promising field of exosome engineering in gene editing, with references to CRISPR technologies and applications that may help make the target exosomes therapeutic in oncology.

Genetic Engineering Pharmaceutical Technology: A Brief Review

Genetic engineering has emerged as a transformative technology in the pharmaceutical industry, offering unprecedented control over the manipulation of genetic material. This technology enables the precise editing, combining, and expression of target genes in various organisms, leading to the production of specific proteins, enzymes, and biologics essential for therapeutic applications. This review provides an overview of the basic principles and strategies of gene therapy, the development of genetic engineering pharmaceuticals, and their applications in producing physiologically active substances, antibodies, and vaccines. The review also highlights the significant advancements in the field, and explores the future potential of genetic engineering in advancing medical science and improving human health.

DNA origami drives gene expression in a human cell culture system

Self-assembling DNA nanoparticles have the potential to significantly advance the targeted delivery of molecular cargo owing to their chemical and architectural flexibility. Recently, it has been demonstrated that the genetic code embedded in DNA nanoparticles produced by the method of DNA origami or related techniques can be recognized and copied by RNA polymerase in vitro. Further, sculpted DNA nanoparticles can serve as a substrate for Cas9-mediated gene modification and gene expression in cell culture. In the present study, we further investigate the ability of DNA origami nanoparticles to be expressed in a human cell line with emphasis on the impact of single-stranded DNA (ssDNA) domains and the contributions of the architectural disposition of genetic control elements, namely promoter and enhancer sequences. Our findings suggest that while cells possess the remarkable capability to express genes within highly folded architectures, the presence and relative density and location of ssDNA domains appears to influence overall levels of gene expression. These results suggest that it may be possible to nuance folded DNA nanoparticle architecture to regulate the rate and/or level of gene expression. Considering the highly malleable architecture and chemistry of self-assembling DNA nanoparticles, these findings motivate further exploration of their potential as an economic nanotechnology platform for targeted gene editing, nucleic acid-based vaccines, and related biotherapeutic applications.

Reducing competition between msd and genomic DNA improves retron editing efficiency

Retrons, found in bacteria and used for defense against phages, generate a unique molecule known as multicopy single-stranded DNA (msDNA). This msDNA mimics Okazaki fragments during DNA replication, making it a promising tool for targeted gene editing in prokaryotes. However, existing retron systems often exhibit suboptimal editing efficiency. Here, we identify the msd gene in Escherichia coli, which encodes the noncoding RNA template for msDNA synthesis and carries the homologous sequence of the target gene to be edited, as a critical bottleneck. Sequence homology causes the msDNA to bind to the msd gene, thereby reducing its efficiency in editing the target gene. To address this issue, we engineer a retron system that tailors msDNA to the leading strand of the plasmid containing the msd gene. This strategy minimizes msd gene editing and reduces competition with target genes, significantly increasing msDNA availability. Our optimized system achieves very high retron editing efficiency, enhancing performance and expanding the potential for in vivo techniques that rely on homologous DNA synthesis.

How Helpful May Be a CRISPR/Cas-Based System for Food Traceability?

Genome editing (GE) technologies have the potential to completely transform breeding and biotechnology applied to crop species, contributing to the advancement of modern agriculture and influencing the market structure. To date, the GE-toolboxes include several distinct platforms able to induce site-specific and predetermined genomic modifications, introducing changes within the existing genetic blueprint of an organism. For these reasons, the GE-derived approaches are considered like new plant breeding methods, known also as New Breeding Techniques (NBTs). Particularly, the GE-based on CRISPR/Cas technology represents a considerable improvement forward biotech-related techniques, being highly sensitive, precise/accurate, and straightforward for targeted gene editing in a reliable and reproducible way, with numerous applications in food-related plants. Furthermore, numerous examples of CRISPR/Cas system exploitation for non-editing purposes, ranging from cell imaging to gene expression regulation and DNA assembly, are also increasing, together with recent engagements in target and multiple chemical detection. This manuscript aims, after providing a general overview, to focus attention on the main advances of CRISPR/Cas-based systems into new frontiers of non-editing, presenting and discussing the associated implications and their relative impacts on molecular traceability, an aspect closely related to food safety, which increasingly arouses general interest within public opinion and the scientific community.

Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis

Pneumocandin B0 (PB0) is a lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB0 production. Our findings reveal that 4 of 10 genes are directly involved in PB0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB0 production, while the absence of glhyp or glhtyC led to the complete loss of PB0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB0 production.

Alzheimer’s disease: A CRISPR/CAS9-mediated therapeutic approach

The degenerative nature of Alzheimer's disease (AD) and its severe effects on cognitive function present a major challenge to worldwide healthcare systems. CRISPR/Cas9, one of the most recent developments in gene-editing technology, has created new opportunities to investigate possible AD treatment approaches. The present state of AD research is reviewed in this paper, along with the possibility of using CRISPR/Cas9-mediated methods to target important genetic elements involved in AD pathogenesis. Through targeted gene editing linked to tau protein malfunction, neuroinflammation, and amyloid-beta accumulation, CRISPR/Cas9 presents a viable approach to altering the molecular course of disease. Additionally, using CRISPR/Cas9 in patient-specific induced pluripotent stem cells (iPSCs) may lead to personalized medicine strategies for the treatment of AD. Issues like delivery strategies, off-target impacts, and moral dilemmas are also covered. All things considered, the application of CRISPR/Cas9 technology to AD research is a fresh and potentially revolutionary strategy for creating targeted treatments for this intricate neurodegenerative illness. For the purpose of treating AD, more preclinical and clinical research is necessary to confirm the security and effectiveness of CRISPR/Cas9-based therapies. With the use of the CRISPR/Cas9 system, precise and effective genome modification is possible, enabling targeted editing of particular genes linked to the pathophysiology of AD. Thanks to this technology, genetic mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP) genes that are linked to family types of AD can be corrected. It is feasible to restore normal protein function and possibly lessen the pathogenic processes that underlie AD by fixing these mutations.

Exploring estrogen antagonism using CRISPR/Cas9 to generate specific mutants for each of the receptors

Endocrine disruptors are chemicals that have been in the spotlight for some time now. Their modulating action on endocrine signaling pathways made them a particularly interesting topic of research within the field of ecotoxicology. Traditionally, endocrine disrupting properties are studied using exposure to suspected chemicals. In recent years, a major breakthrough in biology has been the advent of targeted gene editing tools to directly assess the function of specific genes. Among these, the CRISPR/Cas9 method has accelerated progress across many disciplines in biology. This versatile tool allows to address antagonism differently, by directly inactivating the receptors targeted by endocrine disruptors. Here, we used the CRISPR/Cas9 method to knock out the different estrogen receptors in zebrafish and we assessed the potential effects this generates during development. We used a panel of biological tests generally used in zebrafish larvae to investigate exposure to compounds deemed as endocrine disrupting chemicals. We demonstrate that the absence of individual functional estrogen receptors (Esr1, Esr2b, or Gper1) does affect behavior, heart rate and overall development. Each mutant line was viable and could be grown to adulthood, the larvae tended to be morphologically grossly normal. A substantial fraction (70%) of the esr1 mutants presented severe craniofacial deformations, while the remaining 30% of esr1 mutants also had changes in behavior. esr2b mutants had significantly increased heart rate and significant impacts on craniofacial morphometrics. Finally, mutation of gper1 affected behavior, decreased standard length, and decreased bone mineralization as assessed in the opercle. Although the exact molecular mechanisms underlying these effects will require further investigations in the future, we added a new concept and new tools to explore and better understand the actions of the large group of endocrine disrupting chemicals found in our environment.

Genome Editing of Mammalian Cells Through RNA Transcript-Mediated Homologous Recombination Repair.

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

A quantitative assay for the efficiency of RNA-guided genome editing in plants.

RNA-guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double-strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch-on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20-bp spacer either side to preferentially detect full-deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas-mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4-HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.

Examining the Therapeutic Potential of CRISPR-Cas9 Gene Editing in Spondyloarthritis: Efficacy of TNF Inhibitor Adalimumab

Spondyloarthritis (SpA) encompasses a group of inflammatory rheumatic diseases characterized by axial and peripheral joint involvement, enthesitis, and extra-articular manifestations. Despite advancements in understanding the genetic and immunological underpinnings of SpA, current therapeutic options, including biologic therapies such as the TNF inhibitor adalimumab, primarily focus on symptom management and often show variable efficacy among patients. The advent of CRISPR-Cas9 gene editing technology presents a revolutionary opportunity to directly modify genetic contributors to SpA, potentially transforming the therapeutic landscape and enhancing the efficacy of adalimumab. This review investigates the feasibility and implications of utilizing CRISPR-Cas9 for targeted gene editing in SpA to improve patient response to adalimumab. We review the current knowledge on genetic mutations and polymorphisms associated with SpA pathogenesis, focusing on key genes involved in immune regulation and inflammatory pathways targeted by adalimumab. By leveraging CRISPR-Cas9, we aim to explore how precise genetic modifications can optimize the molecular targets of adalimumab, thereby enhancing its efficacy and reducing refractory cases. Preliminary studies in animal models and cell cultures demonstrate promising results, showcasing the potential of CRISPR-Cas9 to correct genetic abnormalities and synergize with adalimumab to restore normal cellular functions and reduce inflammation. However, translating these findings into clinical applications requires addressing significant challenges, including delivery methods, off-target effects, and long-term safety. Future research directions include optimizing CRISPR-Cas9 delivery systems, such as viral vectors and nanoparticle-based approaches, to enhance specificity and efficiency in conjunction with adalimumab. Additionally, comprehensive preclinical studies and early-phase clinical trials are essential to evaluate the combined therapeutic potential and safety profile of CRISPR-Cas9 and adalimumab in human subjects with SpA. This pioneering approach not only holds the promise of groundbreaking treatments for SpA but also sets the stage for broader applications of gene editing in rheumatology. By targeting the genetic roots of rheumatic diseases and optimizing adalimumab interactions, CRISPR-Cas9 could offer a new era of precision medicine, offering more effective and durable therapeutic outcomes.

The Art of Finding the Right Drug Target: Emerging Methods and Strategies.

Drug targets are specific molecules in biological tissues and body fluids that interact with drugs. Drug target discovery is a key component of drug discovery and is essential for the development of new drugs in areas such as cancer therapy and precision medicine. Traditional in vitro or in vivo target discovery methods are time-consuming and labor-intensive, limiting the pace of drug discovery. With the development of modern discovery methods, the discovery and application of various emerging technologies have greatly improved the efficiency of drug discovery, shortened the cycle time, and reduced the cost. This review provides a comprehensive overview of various emerging drug target discovery strategies, including computer-assisted approaches, drug affinity response target stability, multiomics analysis, gene editing, and nonsense-mediated mRNA degradation, and discusses the effectiveness and limitations of the various approaches, as well as their application in real cases. Through the review of the aforementioned contents, a general overview of the development of novel drug targets and disease treatment strategies will be provided, and a theoretical basis will be provided for those who are engaged in pharmaceutical science research. SIGNIFICANCE STATEMENT: Target-based drug discovery has been the main approach to drug discovery in the pharmaceutical industry for the past three decades. Traditional drug target discovery methods based on in vivo or in vitro validation are time-consuming and costly, greatly limiting the development of new drugs. Therefore, the development and selection of new methods in the drug target discovery process is crucial.

Vascularization of kidney organoids: different strategies and perspectives

Kidney diseases such as glomerulopathy and nephron dysfunction are estimated to grow to more than 900 million cases by 2030, in 45% of which kidney transplantation will be required, representing a major challenge for biomedicine. A wealth of progress has been made to model human diseases using induced pluripotent stem cells (iPSCs) in vitro differentiated to a variety of organoids, including kidney organoids, and in developing various microfluidics-based organ-on-a-chip (OoC) systems based on them. With the combination of targeted gene editing capacities, relevant polymorphic genetic variants can be established in such organoid models to advance evidence-based medicine. However, the major drawback of the current organoid disease models is the lack of functional endothelial vasculature, which especially concerns the kidney, the function of which is strongly associated with blood flow. The design of novel medical devices using tissue engineering approaches such as kidney organoids is also strongly dependent on the understanding of the fundamental principles of nephrogenesis and the vascularization of organs and tissues. Developmental vascularization of the kidney has been an area of intense research for decades. However, there is still no consensus among researchers on how exactly the vascularization of the kidney occurs in normal and pathological conditions. This lack of consensus is partly due to the lack of an appropriate model system to study renal vascularization during nephrogenesis. In this review, we will describe recent progress in the areas of kidney vasculature development, kidney organoids in general and assembled on microfluidic devices in particular. We will focus on the in vitro vasculature of kidney organoids in microfluidic OoC model systems to study kidney diseases and on the perspectives of tissue engineering for the modeling of kidney diseases and the design of bioartificial medical devices. We also aim to summarize the information related to the key mechanisms of intercellular communication during nephrogenesis and the formation of the renal vasculature in an OoC setup.

TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori.

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL-CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL-CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection.

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-colour genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-colour phenotype. Specifically, while young larvae exhibit orange-coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body colour, with Fo-w mutants having a lighter coloured body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene function and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.

An Antibody-CRISPR/Cas Conjugate Platform for Target-Specific Delivery and Gene Editing in Cancer.

The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody-CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2-positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody-conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2-positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody-CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2-positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody-mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases.