CRISPR Research Articles

Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling

Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling

Highly Efficient Homozygous CRISPR/Cas9 Gene Editing Based on Single-Cell-Originated Somatic Embryogenesis in Liriodendron tulipifera

The clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is the most widely used gene-editing tool to date. However, its application in the genetic improvement of forestry trees has been largely limited. Here, we first established a highly efficient multi-target editing system in the magnoliid woody plant Liriodendron tulipifera. Using phytoene desaturase gene (PDS) as an example, we systematically compared CRISPR/Cas9 and CRSPR/Cpf1 expression systems for loss-of-function analysis and conducted genetic transformations using transient and stable transformation. Ultimately, our findings indicated that the CRISPR/Cas9 system, when applied to transformation based on single-cell-originated somatic embryogenesis, yielded the highest gene-editing efficiency, with mutation rates of nearly 100%. Furthermore, we obtained a total of 137 regeneration plantlets via somatic embryogenesis, of which 82.48% exhibited an albino phenotype. The Illumina sequencing results of albino seedlings and the callus tissue obtained from dedifferentiation of mutant plants revealed that the mutation at the T1 target site was homozygous. These results indicate that CRISPR/Cas9-based multiplex genome-editing technology can not only accelerate the identification of gene function but also be incorporated into the genetic improvement and breeding of tulip trees, supporting the scale propagation of genome-edited plantlets via somatic embryogenesis.

Multiplex genome editing for climate-resilient woody plants

Climate change is severely impacting global forest ecosystems, stressing woody plants due to rising temperatures, shifting precipitation patterns, and extreme weather events. These pressures threaten biodiversity and disrupt the essential roles forests play in carbon sequestration, timber production, and ecosystem stability. Traditional forest management strategies, such as selective breeding, cannot keep up with the rapid pace of climate change, given the long juvenile phase of trees. Multiplex genome editing, particularly through CRISPR technologies, offers a promising solution to accelerate the development of climate-resilient traits in woody plants. By simultaneously targeting multiple genes, multiplex CRISPR enables efficient modification of polygenic traits that govern stress tolerance, disease resistance, and other crucial resilience factors. This mini-review examines the potential of multiplex CRISPR technologies in forest management, breeding, and agroecological practices, showing how they can improve tree resilience and support sustainable forestry in response to the growing challenges of climate change.

CRISPR in clinical diagnostics: bridging the gap between research and practice.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has transformed molecular biology through its precise gene-editing capabilities. Beyond its initial applications in genetic modification, CRISPR has emerged as a powerful tool in diagnostics and biosensing. This review explores its transition from genome editing to innovative detection methods, including nucleic acid identification, single nucleotide polymorphism (SNP) analysis, and protein sensing. Advanced technologies such as SHERLOCK and DETECTR demonstrate CRISPR's potential for point-of-care diagnostics, enabling rapid and highly sensitive detection. The integration of chemical modifications, CRISPR-Chip technology, and enzymatic systems like Cas12a and Cas13a enhances signal amplification and detection efficiency. These advancements promise decentralized, real-time diagnostic solutions with significant implications for global healthcare. Furthermore, the fusion of CRISPR with artificial intelligence and digital health platforms is paving the way for more accessible, cost-effective, and scalable diagnostic approaches, ultimately revolutionizing precision medicine.

Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex

Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.

A Comprehensive Review on CRISPR Technology in the Treatment and Understanding of Cardiacmyopathy

A Comprehensive Review on CRISPR Technology in the Treatment and Understanding of Cardiacmyopathy

Functional screen identifies RBM42 as a mediator of oncogenic mRNA translation specificity.

Oncogenic protein dosage is tightly regulated to enable cancer formation but how this is regulated by translational control remains unknown. The Myc oncogene is a paradigm of an exquisitely regulated oncogene and a driver of pancreatic ductal adenocarcinoma (PDAC). Here we use a CRISPR interference screen in PDAC cells to identify activators of selective MYC translation. The top hit, the RNA-binding protein RBM42, is highly expressed in PDAC and predicts poor survival. We show that RBM42 binds and selectively regulates the translation of MYC and a precise suite of pro-oncogenic transcripts, including JUN and EGFR. Mechanistically, we find that RBM42 binds and remodels the MYC 5' untranslated region structure, facilitating the formation of the translation pre-initiation complex. Importantly, RBM42 is necessary for PDAC tumorigenesis in a Myc-dependent manner in vivo. This work transforms the understanding of the translational code in cancer and illuminates therapeutic openings to target the expression of oncogenes.

Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing.

The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies has opened up groundbreaking possibilities for treating a wide spectrum of genetic disorders and diseases. However, the success of these technologies relies heavily on the development of efficient and safe delivery systems. Among the most promising approaches are natural and synthetic nanocarrier-mediated delivery systems, including viral vectors, extracellular vesicles (EVs), engineered cellular membrane particles, liposomes, and various nanoparticles. These carriers enhance the efficacy of the CRISPR system by providing a unique combination of efficiency, specificity, and reduced immunogenicity. Synthetic carriers such as liposomes and nanoparticles facilitate CRISPR delivery with high reproducibility and customizable functions. Viral vectors, renowned for their high transduction efficiency and broad tropism, serve as powerful vehicles for delivering CRISPR components to various cell types. EVs, as natural carriers of RNA and proteins, offer a stealth mechanism to evade immune detection, allowing for the targeted delivery of genome editors with minimal off-target effects. Engineered cellular membrane particles further improve delivery by simulating the cellular environment, enhancing uptake, and minimizing immune response. This review explores the innovative integration of CRISPR genome editors with various nanocarrier systems, focusing on recent advancements, applications, and future directions in therapeutic genome editing.

Isolation of Enterococcus hirae From Fresh White Yak Milk in Ledu District, Qinghai Province, China: A Comparative Genomic Analysis.

Yak milk is a widely consumed dairy product rich in lactic acid bacteria. Although Enterococcus hirae (E. hirae) is commonly found in dairy products and other foods, there is limited information available on its genetic makeup in yak milk. In the present study, 10 E. hirae strains isolated and identified from fresh white yak milk samples, along with 442 E. hirae strains obtained from the NCBI database (totaling 452 strains), were subjected to comparative genomic analysis. The findings of this study revealed that E. hirae has an open pan-genomic structure that allows for its high adaptability and environmental plasticity. Notably, E. hirae isolates from fresh white yak milk had smaller genomes, encoded more functional genes, and had fewer copies of genes encoding carbohydrate-active enzymes involved in the degradation of oligosaccharide metabolism and autolysin synthesis (CE1, GH73, GH23, and GT4 families) than those from animal and human isolates (P < 0.05). Additionally, fresh white yak milk isolates carried only three intrinsic bacteriocins and lacked virulence factors, CRISPR-Cas systems, and resistance genes linked to pathogenicity, which may be attributed to their specialization in the milk-derived environment. This study provides new insights into the genetic and functional gene diversity of E. hirae and how it adapts to milk-derived habitats.

Relationship Between CRISPR–Cas Systems and Acquisition of Tetracycline Resistance in Non-Clinical Enterococcus Populations in Bulgaria

Non-clinical enterococci are relatively poorly studied by means of acquired antibiotic resistance to tetracycline and by the distribution, functionality and role of their CRISPR systems. Background: In our study, 72 enterococcal strains, isolated from various non-clinical origins, were investigated for their phenotypic and genotypic (tet(M), tet(O), tet(S), tet(L), tet(K), tet(T) and tet(W)) tetracycline resistance. Methods: The genetic determinants for HGT (MGEs (Int-Tn and prgW), inducible pheromones (cpd, cop and cff), aggregation substances (agg, asa1, prgB and asa373) and CRISPR–Cas systems were characterized by PCR and whole-genome sequencing. Results: Four tet genes (tetM, tetO, tetS and tetT) were detected in 39% (n = 28) of our enterococcal population, with tetM (31%) being dominant. The gene location was linked to the Tn6009 transposon. All strains that contained tet genes also had genes for HGT. No tet genes were found in E. casseliflavus and E. gilvus. In our study, 79% of all tet-positive strains correlated with non-functional CRISPR systems. The strain E. faecalis BM15 was the only one containing a combination of a functional CRISPR system (cas1, cas2, csn2 and csn1/cas9) and tet genes. The CRISPR subtype repeats II-A, III-B, IV-A2 and VI-B1 were identified among E. faecalis strains (CM4-II-A, III-B and VI-B1; BM5-IV-A2, II-A and III-B; BM12 and BM15-II-A). The subtype II-A was the most present. These repeats enclosed a great number of spacers (1–10 spacers) with lengths of 31 to 36 bp. One CRISPR locus was identified in plasmid (p.Firmicutes1 in strain E. faecalis BM5). We described the presence of CRISPR loci in the species E. pseudoavium, E. pallens and E. devriesei and their lack in E. gilvus, E. malodoratus and E. mundtii. Conclusions: Our findings generally describe the acquisition of foreign DNA as a consequence of CRISPR inactivation, and self-targeting spacers as the main cause.

Characterisation of the CRISPR-Cas systems in Enterococcus faecalis from commercial broiler farm environments and its association with antimicrobial resistance

ABSTRACT 1. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) systems have been highlighted for their potential applications in controlling the spread of mobile genetic elements, including antimicrobial resistance (AMR) genes. This study investigated the characteristics of CRISPR-Cas systems in E. faecalis from commercial broiler farms and assessed the impact of these systems on AMR. 2. All E. faecalis isolates contained CRISPR2, and CRISPR1-Cas and CRISPR3-Cas were identified in 84 (56.4%) and 144 (96.6%) isolates. A combination of CRISPR2 and CRISPR3-Cas and a combination of CRISPR1-Cas, CRISPR2 and CRISPR3-Cas were each identified in 27 (96.4%) farms. 3. There were significant differences between CRISPR-Cas systems for phenotypic AMR: CRISPR1-Cas and CRISPR3-Cas. The E. faecalis isolates without CRISPR1-Cas showed higher resistance to most antimicrobials and had a higher prevalence of multidrug resistance (MDR) than those with CRISPR1-Cas. However, the resistance rate against most antimicrobials and the prevalence of MDR did not differ significantly depending on the presence or absence of CRISPR3-Cas. 4. The E. faecalis isolates without CRISPR1-Cas harboured higher levels of all AMR genes, except for tetL, than those with CRISPR1-Cas. However, the E. faecalis isolates with CRISPR3-Cas showed a significant lower prevalence of tetL gene and a significantly higher prevalence of fexA and poxtA genes. 5. In the distribution of rep families, the rep 9 family was predominant, followed by rep 1, rep 7, rep 2 and rep 8 families. Only prevalence of the rep 7 family was significantly higher in the E. faecalis isolates without CRISPR1-Cas (15.4%) than in those with CRISPR1-Cas (0%). 6. This study is the first report on the characteristics of CRISPR-Cas systems in E. faecalis isolated from commercial broiler farm environments, and the results supported the hypothesis that the development of antimicrobial strategies requires an understanding of the distinctive capabilities between CRISPR1-Cas and CRISPR3-Cas and their underlying resistance mechanisms.

Appraisal of CRISPR Technology as an Innovative Screening to Therapeutic Toolkit for Genetic Disorders.

The high frequency of genetic diseases compels the development of refined diagnostic and therapeutic systems. CRISPR is a precise genome editing tool that offers detection of genetic mutation with high sensitivity, specificity and flexibility for point-of-care testing in low resource environment. Advancements in CRISPR ushered new hope for the detection of genetic diseases. This review aims to explore the recent advances in CRISPR for the detection and treatment of genetic disorders. It delves into the advances like next-generation CRISPR diagnostics like nano-biosensors, digitalized CRISPR, and omics-integrated CRISPR technologies to enhance the detection limits and to facilitate the "lab-on-chip" technologies. Additionally, therapeutic potential of CRISPR technologies is reviewed to evaluate the implementation potential of CRISPR technologies for the treatment of hematological diseases, (sickle cell anemia and β-thalassemia), HIV, cancer, cardiovascular diseases, and neurological disorders, etc. Emerging CRISPR therapeutic approaches such as base/epigenetic editing and stem cells for the development of foreseen CRIPSR drugs are explored for the development of point-of-care testing. A combination of predictive models of artificial intelligence and machine learning with growing knowledge of genetic disorders has also been discussed to understand their role in acceleration of genetic detection. Ethical consideration are briefly discussed towards to end of review. This review provides the comprehensive insights into advances in the CRISPR diagnostics/therapeutics which are believed to pave the way for reliable, effective, and low-cost genetic testing.

Role of CRISPR-Cas System on Virulence traits and Carbapenem Resistance in Clinical Klebsiella pneumoniae Isolates

Background and Objective(s)The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence. Materials and MethodsA total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR. ResultsThe development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW. ConclusionCRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.

Advancements in CRISPR-Based Therapies for Genetic Modulation in Neurodegenerative Disorders.

Neurodegenerative disorders pose significant challenges in the realm of healthcare, as these conditions manifest in complex, multifaceted ways, often attributed to genetic anomalies. With the emergence of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, a new frontier has been unveiled in the quest for targeted, precise genetic manipulation. This abstract explores the recent advancements and potential applications of CRISPR-based therapies in addressing genetic components contributing to various neurodegenerative disorders. The review delves into the foundational principles of CRISPR technology, highlighting its unparalleled ability to edit genetic sequences with unprecedented precision. In addition, it talks about the latest progress in using CRISPR to target specific genetic mutations linked to neurodegenerative diseases like Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Parkinson's disease. It talks about the most important studies and trials that show how well and safely CRISPR-based therapies work. This shows how this technology can change genetic variants that cause diseases. Notably, the discussion emphasizes the challenges and ethical considerations associated with the implementation of CRISPR in clinical settings, including off-target effects, delivery methods, and long-term implications. Furthermore, the article explores the prospects and potential hurdles in the widespread application of CRISPR technology for treating neurodegenerative disorders. It touches upon the need for continued research, improved delivery mechanisms, and ethical frameworks to ensure responsible and equitable access to these groundbreaking therapies.

A magnetic bead-based dual-aptamer sandwich assay for quantitative detection of ciprofloxacin using CRISPR/Cas12a.

Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antibiotic, and its excessive residues in food and water sources pose potential risks to human health. Therefore, there is a need for a rapid and convenient method for its accurate quantification. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system has gained extensive application in signal detection and amplification due to the trans-cleavage activity of Cas12a. In this study, we devised a novel magnetic bead-based dual sandwich aptamer coupled with a CRISPR/Cas12a system for the precise quantification of CIP in milk, river water, and honey. Through the incorporation of a magnetic bead-based dual aptamer sandwich approach, the concentration of CIP in the samples was pre-enriched. Additionally, by optimizing the Fluorescence-Quencher (F-Q) probe concentration, detection aptamer (APTd) concentration, and assay duration, the limit of blank (LOB) of the system was determined as 362nM, while the limit of detection (LOD) was determined as 403nM. This enabled the accurate quantification of CIP within the linear range of 0.5μM to 0.2mM with high specificity. Moreover, the performance of this detection method was comparable to that of high-performance liquid chromatography (HPLC) in river water, milk, and honey samples.

Recent advances in lateral flow assays for MicroRNA detection.

Lateral flow assays (LFAs) have emerged as pivotal tools for the rapid and reliable detection of microRNAs (miRNAs). It is believed that these biomarkers are crucial for the diagnosis and prognosis of various diseases, particularly cancer. Traditional miRNA detection techniques, such as quantitative PCR, are highly sensitive but have limited efficacy due to their complexity, high cost, and technical requirements. LFAs are valuable due to their simplicity, affordability, and portability, making them ideal for point-of-care testing in low-resource environments. However, challenges remain in developing highly sensitive and accurate LFA devices for miRNA detection. This review explores recent advancements in LFAs to improve miRNA detection sensitivity and specificity. Key innovations include signal amplification using isothermal methods, the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems for direct targeting of miRNAs, and the incorporation of nanomaterials, such as gold nanoparticles and nanorods, to enhance signal intensity. Using artificial intelligence (AI) algorithms enables precise, automated, and rapid quantification of miRNAs. Moreover, this review examines the ability of LFA-based devices to detect multiple miRNAs simultaneously. One of the most significant advancements is the detection of miR-21 levels as low as 20 pM and let-7a levels as low as 40 pM within ten minutes. This highlights the potential of these devices for clinical diagnostics.

Focused ultrasound widely broadens AAV-delivered Cas9 distribution and activity.

Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.

Photosynthesis in Synechocystis sp. PCC 6803 is not optimally regulated under very high CO2

One strategy for CO2 mitigation is using photosynthetic microorganisms to sequester CO2 under high concentrations, such as in flue gases. While elevated CO2 levels generally promote growth, excessively high levels inhibit growth through uncertain mechanisms. This study investigated the physiology of the cyanobacterium Synechocystis sp. PCC 6803 under very high CO2 concentrations and yet stable pH around 7.5. The growth rate of the wild type (WT) at 200 µmol photons m−2 s−1 and a gas phase containing 30% CO2 was 2.7-fold lower compared to 4% CO2. Using a CRISPR interference mutant library, we identified genes that, when repressed, either enhanced or impaired growth under 30% or 4% CO2. Repression of genes involved in light harvesting (cpc and apc), photochemical electron transfer (cytM, psbJ, and petE), and several genes with little or unknown functions promoted growth under 30% CO2, while repression of key regulators of photosynthesis (pmgA) and CO2 capture and fixation (ccmR, cp12, and yfr1) increased growth inhibition under 30% CO2. Experiments confirmed that WT cells were more susceptible to light inhibition under 30% than under 4% CO2 and that a light-harvesting-impaired ΔcpcG mutant showed improved growth under 30% CO2 compared to the WT. These findings suggest that enhanced fitness under very high CO2 involves modifications in light harvesting, electron transfer, and carbon metabolism, and that the native regulatory machinery is insufficient, and in some cases obstructive, for optimal growth under 30% CO2. This genetic profiling provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience for biotechnological applications.Key points• Synechocystis growth was inhibited under very high CO2.• Inhibition of growth under very high CO2 was light dependent.• Repression of photosynthesis genes improved growth under very high CO2.Graphical

Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.

Cas12a is a next-generation geneediting tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed geneediting in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo geneediting, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.

From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.

Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.