CRISPR Research Articles

Multi-dimensional bio mass cytometry: simultaneous analysis of cytoplasmic proteins and metabolites on single cells.

Single-cell multi-dimensional analysis enables more profound biological insight, providing a comprehensive understanding of cell physiological processes. Due to limited cellular contents, the lack of protein and metabolite amplification ability, and the complex cytoplasmic environment, the simultaneous analysis of intracellular proteins and metabolites remains challenging. Herein, we proposed a multi-dimensional bio mass cytometry platform characterized by protein signal conversion and amplification through an orthogonal exogenous enzymatic reaction. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology was applied in the quantification of endogenous intracellular protein glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) through exogenous luciferase Nanoluc (Nluc). The simultaneous detection of GAPDH and hundreds of metabolites at the single-cell level was realized for the first time. Semiquantitative analysis of GAPDH together with single-cell metabolomes under S-nitrosoglutathione (GSNO)-induced oxidative stress was investigated. Bioinformatics analysis revealed 16 metabolites that correlated positively with GAPDH expression upon oxidative stress, including long-chain fatty acids (palmitoleic acid, myristic acid, etc.) and UDP-N-acetylglucosamine (UDP-GlcNAc). Potential synergetic functions of GAPDH and UDP-GlcNAc-mediated oxidative stress responses were also elucidated. Our work proposes a novel strategy for the simultaneous quantitative analysis of single-cell intracellular proteins and metabolites, deepens the understanding of inherent anti-oxidative stress response mechanisms, and provides the molecular fundamentals for the study of inherent biological processes.

The Role of M1 and M2 in Modulating Ab Accumulation and Function

Alzheimer's disease is a neurodegenerative disorder characterized by progressive cognitive decline, memory loss, and the formation of amyloid-beta plaques and tau tangles in the brain. This chronic condition has no known cure, and its progression varies greatly between individuals, eventually leading to severe cognitive impairment, loss of independence, and death. Microglia, the primary immune cells in the central nervous system that serve as the brain's first line of defense, are central to Alzheimer's disease pathology. Microglia play an important role in the response to injury and infection, as well as the regulation of amyloid-beta levels. However, when amyloid-beta accumulates, it activates an inflammatory response via microglia. While this response is initially protective, it may become chronic, contributing to the neuroinflammation that worsens Alzheimers disease pathology. In this study, I wanted to isolate and investigate the specific effects of M1 and M2 macrophages in the brains of genetically modified mice. Using CRISPR technology, we developed mouse models with selective expression of M1 macrophages without M2, M2 macrophages without M1, and models with both macrophage types. Western blot analysis quantified the levels of amyloid-beta in these mice, revealing how microglial activity influences Alzheimer's disease progression. Our findings show that M1 macrophages primarily regulate amyloid-beta through inflammatory processes, which may increase amyloid-beta production, whereas M2 macrophages are essential for amyloid-beta clearance. These findings emphasize the importance of balancing M1 and M2 macrophage activity when treating Alzheimer's disease

CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds.

Forkhead box L2 (FOXL2) encodes a transcription factor essential for sex determination, and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation and invasion. Furthermore, these modified cells are more susceptible to dasatinib and ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type-like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing GCTs, highlighting its therapeutic promise for treating this rare ovarian cancer.

Modulation of CRISPR-Cas9 Cleavage with an Oligo-Ribonucleoprotein Design.

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein Cas9 system has been widely used for genome editing. However, the editing or cleavage specificity of CRISPR Cas9 remains a major concern due to the off-target effects. The existing approaches to control or modulate CRISPR Cas9 cleavage include engineering Cas9 protein and development of anti-CRISPR proteins. There are also attempts on direct modification of sgRNA, for example, structural modification via truncation or hairpin design, or chemical modification on sgRNA such as partially replacing RNA with DNA. The above-mentioned strategies rely on extensive protein engineering and direct chemical or structural modification of sgRNA. In this study, we proposed an indirect method to modulate CRISPR Cas9 cleavage without modification on Cas9 protein or sgRNA. An oligonucleotide was used to form an RNA-DNA hybrid structure with the sgRNA spacer, creating steric hindrance during the Cas9 mediated DNA cleavage process. We first introduced a simple and robust method to assemble the oligo-ribonucleoprotein (oligo-RNP). Next, the cleavage efficiency of the assembled oligo-RNP was examined using different oligo lengths in vitro. Lastly, we showed that the oligo-RNP directly delivered into cells could also modulate Cas9 activity inside cells using three model gene targets with reduced off-target effects.

CRISPR/Cas System: A Powerful Strategy to Improve Monogenic Human Diseases as Therapeutic Delivery; Current Applications and Challenges.

The 5,000 to 8,000 monogenic diseases are inherited disorders leading to mutations in a single gene. These diseases usually appear in childhood and sometimes lead to morbidity or premature death. Although treatments for such diseases exist, gene therapy is considered an effective and targeted method and has been used in clinics for monogenic diseases since 1989. Monogenic diseases are good candidates for novel therapeutic technologies like gene editing approaches to repair gene mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, the pioneer and effective gene editing tool, are utilized for ex vivo and in vivo treatment of monogenic diseases. The current review provides an overview of recent therapeutic applications of CRISPR-based gene editing in monogenic diseases in in vivo and ex vivo models. Furthermore, this review consolidates strategies aimed at providing new treatment options with gene therapy, thereby serving as a valuable reference for advancing the treatment landscape for patients with monogenic disorders.

Trends in Aptasensing and the Enhancement of Diagnostic Efficiency and Accuracy.

The field of healthcare diagnostics is navigating complex challenges driven by evolving patient demographics and the rapid advancement of new technologies worldwide. In response to these challenges, these biosensors offer distinctive advantages over traditional diagnostic methods, such as cost-effectiveness, enhanced specificity, and adaptability, making their integration with point-of-care (POC) platforms more feasible. In recent years, aptasensors have significantly evolved in diagnostic capabilities through the integration of emerging technologies such as microfluidics, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, wearable devices, and machine learning (ML), driving progress in precision medicine and global healthcare solutions. Moreover, these advancements not only improve diagnostic accuracy but also hold the potential to revolutionize early detection, reduce healthcare costs, and improve patient outcomes, especially in resource-limited settings. This Account examines key advancements, focusing on how scientific breakthroughs, including artificial intelligence (AI), have improved sensitivity and precision. Additionally, the integration of aptasensors with these technologies has enabled real-time monitoring and data analysis, fostering advances in personalized healthcare. Furthermore, the potential commercialization of aptasensor technologies could increase their availability in clinical settings and support their use as widespread solutions for global health challenges. Hence, this review discusses technological improvements, practical uses, and prospects while also focusing on the challenges surrounding standardization, clinical validation, and interdisciplinary collaboration for widespread application. Finally, ongoing efforts to address these challenges are key to ensure that aptasensors can be effectively implemented in diverse healthcare systems.

Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.

CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model trained on a dataset of over 45,000 CRISPR-Cas PAMs. Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across Type I, II, and V CRISPR-Cas systems. Using in silico deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild-type under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.

Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.

Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.

Gene Editing: Developments, Ethical Considerations, and Future Directions.

An examination of recent developments related to CRISPR technology, ethical considerations of the application of such technologies, and future directions for germline editing.

Leveraging Synthetic Antibody-DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox.

We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.

Breast Cancer Subtypes and Current Promising Genetic Engineering Tools for Breast Cancer Treatment - An Overview

Abstract: Breast cancer poses a significant global health challenge, and if current trends persist, the burden of breast cancer is projected to escalate, yielding over 3 million new cases and 1 million fatalities annually by the year 2040. Breast cancer is a highly heterogeneous disease, presenting a spectrum of subtypes, each characterized by unique clinical behaviors and responses to treatments. Understanding these breast cancer subtypes is of paramount importance in the fields of oncology and personalized medicine. In addition to conventional breast cancer treatments, such as surgery, chemotherapy, radiotherapy, hormonal therapy, and immunotherapy, recent scientific advancements have introduced a range of genetic engineering tools with noteworthy potential. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR), and small interfering RNA (siRNA) have emerged as promising components of breast cancer treatment. These tools offer encouraging applications due to their precision in targeting and manipulating genes. This review presents a comprehensive exploration of the various subtypes of breast cancer, along with an examination of the current promising genetic engineering tools in treating breast cancer. It sheds light on their roles in the evolving landscape of breast cancer treatment.

DCas-Based Tools to Visualize Chromatin or Modify Epigenetic Marks at Specific Plant Genomic Loci.

Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.

Optimizing Crop Production With Plant Phenomics Through High‐Throughput Phenotyping and AI in Controlled Environments

ABSTRACTPlant phenomics deals with the measurement of plant phenotypes associated with genetic and environmental variation in controlled environment agriculture (CEA). Encompassing a spectrum from molecular biology to ecosystem‐level studies, it employs high‐throughput phenotyping (HTP) approaches to quickly evaluate characteristics and enhance the yields of crops in smart plant facilities. HTP uses environmental parameters for accuracy, such as software sensors, as well as hyperspectral imaging for pigment data, thermal imaging for water content, and fluorescence imaging for photosynthesis rates. They provide information on growth kinetics, physiological and biochemical characteristics, and genotype–environment interaction. Artificial intelligence (AI) and machine learning (ML) are used on a large volume of phenotypic data to predict growth rates, determine the optimal time to water plants, or detect diseases, nutrient deficiencies, or pests at an early stage. The lighting used in smart plant factories is adjusted based on the specific growth phase of the plants, such as using different light intensities, spectrums, and durations for germination, vegetative growth, and flowering stages, hydroponics as the method of providing nutrients, and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) for improving certain characteristics, such as resistance to drought. These systems enhance crop production, yields, adaptability, and input use by optimizing the environment and utilizing precision breeding techniques. Plant phenomics with AI is a combination of several disciplines, promoting the understanding of plant–environment interactions in relation to agriculture problems such as resource use, diseases, and climate change. It affects their capacity to develop crops that capture inputs, minimize chemical application, and are resilient to climate change. Phenomics is cost‐effective, reduces inputs, and contributes to more sustainable agricultural practices, being economically and environmentally sound. Altogether, plant phenomics is central to CEA due to its capacity to capitalize on phenotypic data and genetic potential within agriculture to advance sustainability and food security. Through phenomic research, the next advancements are likely to be even more revolutionary in terms of agricultural practices and food systems worldwide.

Molecular characterization and genome sequencing of selected highly resistant clinical isolates of Pseudomonas aeruginosa and its association with the clustered regularly interspaced palindromic repeat/Cas system.

The presence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in the superbug Pseudomonas (P) aeruginosa presents a unique opportunity to precisely target and edit bacterial genomes to modify their drug resistance. The objective was to detect the prevalence of CRISPR in extensively and pan-drug-resistant Pseudomonas aeruginosa and to determine the utility of whole-genome sequencing (WGS) for the analysis of the entire genome for such strains. The antimicrobial susceptibilities of one hundred isolates were assessed using the antibiotic susceptibility test (AST) card of the VITEK system. The presence of the CRISPR/Cas system was determined via specific primers using conventional polymerase chain reaction (PCR). Further, WGS was conducted using a DNA nanoball sequencing platform via BGI-Tech for the isolates of interest. Out of 54 resistant Pseudomonas aeruginosa isolates, 33 (33.0%) were metallo-β-lactamase producers. Cas1, Cas3, CRISPR1, and CRISPR2 were positive in 6.0% of isolates, while incomplete CRISPR1-Cas systems alone were found in 15.0%. Also, CRISPR2-type was found intact in 26% of isolates. The prevalence of resistance to antimicrobials in P. aeruginosa isolates was significantly greater in the CRISPR/Cas-negative group compared to the CRISPR/Cas-positive. Significant relationships for variables were examined using Fisher's exact tests using Chi-squared and a P-value of <0.05 as a statistical threshold. Further, on examination of CRs as a collective entity, encompassing both extensive drug resistance (XDR) and pan-drug resistance (PDR), it becomes evident that the vast majority of these strains (n=29; 87.8%) lacked CRISPR/Cas systems. In phylogenic analysis, PDR-P. aeruginosa revealed a very close evolutionary relationship with those originating from Kazakhstan, while XDR was globally unique. Further, the entire genome showed the presence of unique virulence and resistant pseudomonal genes. The CRISPR/Cas system and drug resistance are antagonistic to one another. XDR and PDR P. aeruginosa represent a potential threat to public health and contribute to the seriousness of associated illnesses by leading to resistant infections. Further, WGS for the two strains revealed resistance to multiple antibiotics. It is important to examine specific antimicrobial resistance (AMR) pathways, which suggests that a significant number of resistant genes in these isolates indicate a loss of CRISPR genes in the two strains. Furthermore, the WGS approach can lead to a better understanding of the genomic mechanism of pseudomonal resistance to antibiotics.

Structure-switchable branched inhibitors regulate the activity of CRISPR-Cas12a for nucleic acid diagnostics

In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot. To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology. The construction-transferable branched inhibitors coupled with a specific overhang flap induce spatial steric effects and result in the loss of the binding ability of Cas12a, which inhibits the activity of Cas12a. Target as the input signal would trigger the site-directed APE1 enzyme incision of the inhibitors, thus transforming the conformation of the inhibitors into split activators to illumine the CRISPR/AsCas12a catalyst system. At the same time, we found that APE1 could drive the enzymatic positive feedback circuit and exhibited considerably high amplification efficiency to enhance the detection ability of nucleic acids. Besides, our method provides universal platforms and can be realized in real-time and one-pot detection of HIV-1 DNA by replacing the inhibitors and crRNA with different target recognition sequences. Overall, due to the high programmability of the nucleic acid network, this work proposed a feasible way to use the steric hindrance-based inhibitors as a switchable element, decorating the CRISPR/Cas12a-based strategy equipment for molecular diagnostics. Besides, this strategy could offer a simple tool for detecting trace nucleic acid, which opens avenues for future clinical application.

Cas12a is competitive for gene editing in the malaria parasites.

Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.

CRISPR/Cas Systems as Diagnostic and Potential Therapeutic Tools for Enterohemorrhagic Escherichia coli.

Following its discovery as an adaptive immune system in prokaryotes, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been developed into a multifaceted genome editing tool. This review compiles findings aimed at implementation of this technology for selective elimination or attenuation of enterohemorrhagic Escherichia coli (EHEC). EHEC are important zoonotic foodborne pathogens that cause hemorrhagic colitis and can progress to the life-threatening hemolytic uremic syndrome (HUS). Advancements in the application of CRISPR methodology include laboratory detection and identification of EHEC, genotyping, screening for pathogenic potential, and engineering probiotics to reduce microbial shedding by cattle, the primary source of human infection. Genetically engineered phages or conjugative plasmids have been designed to target and inactivate genes whose products are critical for EHEC virulence.

Effect of neutral electrolyzed water on biofilm formed by meat-related Listeria monocytogenes: Intraspecies variability and influence of the growth surface material.

Listeria monocytogenes raises major challenges for the food industry. Due to its capacity to form biofilms, this pathogen can persist in processing environments and contaminate the final products. Neutral electrolyzed water (NEW) may offer a promising and eco-friendly method for controlling L. monocytogenes biofilms, though current in vitro studies on its antibiofilm activity are limited and often focused on reference strains. In this study, we assessed the effect of NEW on biofilms formed by meat-related and reference L. monocytogenes strains on polystyrene and stainless steel. Forty wild-type strains isolated from meat products and processing environments were firstly screened for their biofilm-forming abilities and classified as weak (30%; 12/40), moderate (55%; 22/40), and strong (15%; 6/40) biofilm producers. Twenty-two wild-type and two reference strains were selected for the eradication assays, performed by treating the biofilms with NEW for 9minutes of total contact time. In silico functional enrichment analysis and the visualization of biofilms by scanning electron microscopy (SEM) were also performed. The NEW treatment resulted in a greater average reduction of viable cells in biofilms formed on polystyrene (4.3±1.0 log10 CFU/cm2) compared to stainless steel (2.9±2.0 log10 CFU/cm2), and a remarkable intraspecies variability was observed. SEM images revealed higher structural damage on biofilms formed on polystyrene. Functional enrichment analysis suggested that clustered regularly interspaced short palindromic repeats (CRISPR)-associated elements could be involved in resistance to the treatments. NEW could be a promising additional tool to mitigate L. monocytogenes biofilms in meat processing environments, although its effect varied with surface material and strain-specific characteristics.

DNA nanotechnology-based strategies for minimising hybridisation-dependent off-target effects in oligonucleotide therapies.

Targeted therapy has emerged as a transformative breakthrough in modern medicine. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have made significant advancements in targeted therapy. Other oligonucleotide-based therapeutics like clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are also leading a revolution in targeted gene therapy. However, hybridisation-dependent off-target effects, arising from imperfect base pairing, remain a significant and growing concern for the clinical translation of oligonucleotide-based therapeutics. These mismatches in base pairing can lead to unintended steric blocking or cleavage events in non-pathological genes, affecting the efficacy and safety of the oligonucleotide drugs. In this review, we examine recent developments in oligonucleotide-based targeted therapeutics, explore the factors influencing sequence-dependent targeting specificity, and discuss the current approaches employed to reduce the off-target side effects. The existing strategies, such as chemical modifications and oligonucleotide length optimisation, often require a trade-off between specificity and binding affinity. To further address the challenge of hybridisation-dependent off-target effects, we discuss DNA nanotechnology-based strategies that leverage the collaborative effects of nucleic acid assembly in the design of oligonucleotide-based therapies. In DNA nanotechnology, collaborative effects refer to the cooperative interactions between individual strands or nanostructures, where multiple bindings result in more stable and specific hybridisation behaviour. By requiring multiple complementary interactions to occur simultaneously, the likelihood of unintended partially complementary binding events in nucleic acid hybridisation should be reduced. And thus, with the aid of collaborative effects, DNA nanotechnology has great promise in achieving both high binding affinity and high specificity to minimise the hybridisation-dependent off-target effects of oligonucleotide-based therapeutics.

Advances in applications of the CRISPR/Cas9 system for respiratory diseases.

Genetic and environmental factors can have an impact on lung and respiratory disorders which are associated with severe symptoms and have high mortality rates. Many respiratory diseases are significantly influenced by genetic or epigenetic factors. Gene therapy offers a powerful approach providing therapeutic treatment for lung diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) are promising gene modifying tool that can edit the genome. The utilization of CRISPR/Cas9 systems in the investigation of respiratory disorders has resulted in advancements such as the rectification of deleterious mutations in patient-derived cells and the alteration of genes in multiple mammalian lung disease models. New avenues of treatment for lung disorders have been opened up by advances in CRISPR/Cas9 research. In this chapter, we discuss the known genes and mutations that cause several common respiratory disorders such as COPD, asthma, IPF, and ARDS. We further review the current research using CRISPR/Cas9 in numerous respiratory disorders and possible therapeutic treatments.