CRISPR Research Articles

Systematic identification of post-transcriptional regulatory modules

In our cells, a limited number of RNA binding proteins (RBPs) are responsible for all aspects of RNA metabolism across the entire transcriptome. To accomplish this, RBPs form regulatory units that act on specific target regulons. However, the landscape of RBP combinatorial interactions remains poorly explored. Here, we perform a systematic annotation of RBP combinatorial interactions via multimodal data integration. We build a large-scale map of RBP protein neighborhoods by generating in vivo proximity-dependent biotinylation datasets of 50 human RBPs. In parallel, we use CRISPR interference with single-cell readout to capture transcriptomic changes upon RBP knockdowns. By combining these physical and functional interaction readouts, along with the atlas of RBP mRNA targets from eCLIP assays, we generate an integrated map of functional RBP interactions. We then use this map to match RBPs to their context-specific functions and validate the predicted functions biochemically for four RBPs. This study provides a detailed map of RBP interactions and deconvolves them into distinct regulatory modules with annotated functions and target regulons. This multimodal and integrative framework provides a principled approach for studying post-transcriptional regulatory processes and enriches our understanding of their underlying mechanisms.

To what extent is transgenic technology beneficial to human beings?

Genetic engineering is a field which explores the possibility of modification or manipulation of genetic materials using various techniques and technologies. Broadly speaking, hybridisation, gene isolation and recombination, fall in this field. The most recent milestone development in this area would be CRISPR technology, which harnesses a component of the bacterial immune system. To elaborate, CRISPR acts as a precise molecular scissors to cut target sequences, thus allowing gene modification to take place. In the progression and advancement of this field, society has been carefully observing its consequences and impact. The utilisation of this technology has raised concerns such as the unknown detrimental impact of genetically modified food sources. On the other hand, the benefit of genetic engineering is self-evident. This article explores the various impacts of genetic engineering and concludes that the advantages of genetic engineering are overshadowed by its many benefits.

Genetic engineering in oncology based on CRISPR-Cas9 technology

Purpose of the study: analysis of modern scientific data on the molecular mechanisms of the CRISPR-Cas9 system in gene editing, advantages and disadvantages in cancer research and the development of new treatment methods. Material and Methods. A comprehensive electronic search of relevant published studies was conducted in the scientific databases PubMed/MEDLINE, ScienceDirect, Wiley and Google Scholar published between 2014 and 2024. The search was tailored to the specific requirements of each database based on the following keywords: CRISPR-Cas9, sgRNA, genome editing, cancer immunotherapy, CAR-T. The search yielded 487 studies on the topic of interest, of which 54 were used to write the literature review. Additionally, the article discretely highlights the importance and challenges of CRISPR-Cas9 in the production of genetically engineered T cells for potential use in treating certain types of cancer. Results. Accordingly, CAR-T (chimeric antigen receptor T-cell) therapy is widely used as one of the main components of immunotherapy in the treatment of leukemia, lymphoma and some solid tumors. The development of programmed single guide RNAs (sgRNAs) and new modifications of the Cas9 protein has made the technology flexible and universal. CRISPR-Cas9 is often used to modify T and NK cells by designing antigen receptors to improve their sensory circuits with complex functionality capable of recognizing and killing tumor cells. At the same time, delivery of the finished ribonucleoprotein (Cas9+sgRNA) complex into the cell avoids the constitutive processes of transcription and translation, which ensures the fastest possible gene editing. Conclusion. In this review, we reviewed the scientific evidence highlighting the promising impact of CRISPR technologies in cancer research and treatment. CRISPR-Cas9 is considered a unique and effective technology in the field of genetic and biomolecular engineering.

The role of the hepatitis B virus genome and its integration in the hepatocellular carcinoma.

The integration of Hepatitis B Virus (HBV) is now known to be closely associated with the occurrence of liver cancer and can impact the functionality of liver cells through multiple dimensions. However, despite the detailed understanding of the characteristics of HBV integration and the mechanisms involved, the subsequent effects on cellular function are still poorly understood in current research. This study first systematically discusses the relationship between HBV integration and the occurrence of liver cancer, and then analyzes the status of the viral genome produced by HBV replication, highlighting the close relationship and structure between double-stranded linear (DSL)-HBV DNA and the occurrence of viral integration. The integration of DSL-HBV DNA leads to a certain preference for HBV integration itself. Additionally, exploration of HBV integration hotspots reveals obvious hotspot areas of HBV integration on the human genome. Virus integration in these hotspot areas is often associated with the occurrence and development of liver cancer, and it has been determined that HBV integration can promote the occurrence of cancer by inducing genome instability and other aspects. Furthermore, a comprehensive study of viral integration explored the mechanisms of viral integration and the internal integration mode, discovering that HBV integration may form extrachromosomal DNA (ecDNA), which exists outside the chromosome and can integrate into the chromosome under certain conditions. The prospect of HBV integration as a biomarker was also probed, with the expectation that combining HBV integration research with CRISPR technology will vigorously promote the progress of HBV integration research in the future. In summary, exploring the characteristics and mechanisms in HBV integration holds significant importance for an in-depth comprehension of viral integration.

CRISPRi with barcoded expression reporters dissects regulatory networks in human cells.

Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.

The application and reflection of emerging molecular detection technologies in Salmonella detection

Salmonella is an important foodborne pathogen and one of the main causes of diarrhea. Every year, about 550 million people suffer from diarrhea due to Salmonella infection, of which about 230 000 die. It has become a major global public safety issue. The application fields of Salmonella detection involve food safety, water quality monitoring, animal husbandry, public health monitoring, and medical diagnosis. The detection requirements mainly come from three aspects: pathogen identification, serotype identification, drug resistance and virulence identification. In recent years, the detection technology for Salmonella has made rapid progress, especially the emergence and development of emerging molecular detection technologies, providing new perspectives for Salmonella detection in different scenarios. However, due to the diversity of Salmonella serotypes and the complexity of detection scenarios, existing detection technologies still have some pain points (such as long detection time, cumbersome operation steps, low scene adaptability, etc.). This article will elaborate on the application of several emerging molecular detection technologies with distinct characteristics, such as CRISPR Cas technology, digital PCR technology, sequencing technology, and microfluidic technology, in Salmonella detection. It aims to provide a reference for the development and improvement of Salmonella detection technology and the establishment of infection warning and control systems.

An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis

BackgroundThe type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2−based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.ResultsThis study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5′end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.ConclusionsTherefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

Global marine microbial diversity and its potential in bioprospecting

The past two decades has witnessed a remarkable increase in the number of microbial genomes retrieved from marine systems1,2. However, it has remained challenging to translate this marine genomic diversity into biotechnological and biomedical applications3,4. Here we recovered 43,191 bacterial and archaeal genomes from publicly available marine metagenomes, encompassing a wide range of diversity with 138 distinct phyla, redefining the upper limit of marine bacterial genome size and revealing complex trade-offs between the occurrence of CRISPR–Cas systems and antibiotic resistance genes. In silico bioprospecting of these marine genomes led to the discovery of a novel CRISPR–Cas9 system, ten antimicrobial peptides, and three enzymes that degrade polyethylene terephthalate. In vitro experiments confirmed their effectiveness and efficacy. This work provides evidence that global-scale sequencing initiatives advance our understanding of how microbial diversity has evolved in the oceans and is maintained, and demonstrates how such initiatives can be sustainably exploited to advance biotechnology and biomedicine.

Role of CRISPR-Cas systems in periodontal disease pathogenesis and potential for periodontal therapy: A review.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA sequences capable of editing a host genome sequence. CRISPR and its specific CRISPR-associated (Cas) protein complexes have been adapted for various applications. These include activating or inhibiting specific genetic sequences or acting as molecular scissors to cut and modify the host DNA precisely. CRISPR-Cas systems are also naturally present in many oral bacteria, where they aid in nutrition, biofilm formation, inter- and intraspecies communication (quorum sensing), horizontal gene transfer, virulence, inflammation modulation, coinfection, and immune response evasion. It even functions as an adaptive immune system, defending microbes against invading viruses and foreign genetic elements from other bacteria by targeting and degrading their DNA. Recently, CRISPR-Cas systems have been tested as molecular editing tools to manipulate specific genes linked with periodontal disease (such as periodontitis) and as novel methods of delivering antimicrobial agents to overcome antimicrobial resistance. With the rapidly increasing role of CRISPR in treating inflammatory diseases, its application in periodontal disease is also becoming popular. Therefore, this review aims to discuss the different types of CRISPR-Cas in oral microbes and their role in periodontal disease pathogenesis and precision periodontal therapy.

Rice transcription factor DPF regulates stress-induced biosynthesis of diterpenoid phytoalexins.

Diterpenoid Phytoalexin Factor (DPF) is a key transcription factor involved in diterpenoid phytoalexin (DP) biosynthesis under non-stressed conditions in rice (Oryza sativa L.). Using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9, DPF knockout rice lines were generated. Treatments with abiotic stresses (copper chloride, ultraviolet light, and jasmonic acid) and biotic stress (blast fungus infection) to the knockout lines revealed that the DPF positively regulates stress-induced DP biosynthesis.

Application of CRISPR/Cas9 system to knock out GluB gene for developing low glutelin rice mutant

The nutritional quality improvement is among the most integral objective for any rice molecular breeding programs. The seed storage proteins (SSPs) have greater role to determine the nutritional quality of any cereal grains. Rice contains relatively balanced amino acid composition and the SSPs are fractioned into albumins (ALB), globulins (GLO), prolamins (PRO) and glutelins (GLU) according to differences in solubility. GLUs are further divided into subfamilies: GluA, GluB, GluC, and GluD depending on resemblance in amino acid. The GLU protein accounts for 60–80% of total protein contents, encoded by 15 genes located on different chromosomes of rice genome. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was employed to knockout Glu-B (LOC-Os02g15070) gene in non-basmati rice PK386 cultivar. The mutant displayed two base pair and three base pair mutation in the targeted regions. The homozygous mutant plant displayed reduction for both in total protein contents and GLU contents whereas, elevation in GLO, ALB and PRO. Moreover, the mutant plant also displayed reduction in physio-chemical properties e.g., total starch, amylose and gel consistency. The agronomic characteristics of both mutant and wild type displayed non-significant differences along with increase in higher percentage of chalkiness in mutant plants. The results obtained from scanning electron microscopy showed the loosely packed starch granules compared to wild type. The gene expression analysis displayed the lower expression of gene at 5 days after flowering (DAF), 10 DAF, 15 DAF and 20 DAF compared to wild type. GUS sub-cellular localization showed the staining in seed which further validated the results obtained from gene expression. Based on these findings it can be concluded Glu-B gene have significant role in controlling GLU contents and can be utilized in breeding programs to enhance the nutritional quality of rice, and may serve as healthy diet for patient allergic with high GLU contents.

New Viruses Infecting Hyperthermophilic Bacterium Thermus thermophilus.

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.

Genome editing for improvement of biotic and abiotic stress tolerance in cereals

Global agricultural production must quadruple by 2050 to fulfil the needs of a growing global population, but climate change exacerbates the difficulty. Cereals are a very important source of food for the world population. Improved cultivars are needed, with better resistance to abiotic stresses like drought, salt, and increasing temperatures, and resilience to biotic stressors like bacterial and fungal infections, and pest infestation. A popular, versatile, and helpful method for functional genomics and crop improvement is genome editing. Rapidly developing genome editing techniques including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) are very important. This review focuses on how CRISPR/Cas9 genome editing might enhance cereals’ agronomic qualities in the face of climate change, providing important insights for future applications. Genome editing efforts should focus on improving characteristics that confer tolerance to conditions exacerbated by climate change (e.g. drought, salt, rising temperatures). Improved water usage efficiency, salt tolerance, and heat stress resilience are all desirable characteristics. Cultivars that are more resilient to insect infestations and a wide range of biotic stressors, such as bacterial and fungal diseases, should be created. Genome editing can precisely target genes linked to disease resistance pathways to strengthen cereals’ natural defensive systems.

Deciphering regulation of FOXP3 expression in human conventional T cells.

FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation.

Trafficking of G protein-coupled receptors (GPCRs) through the endosomal-lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and the large number of cellular proteins involved in the process. Here, we developed a high-sensitivity biosensor for GPCR expression and agonist-induced trafficking to the lysosome by leveraging the ability of the engineered peroxidase APEX2 to activate the fluorogenic substrate Amplex UltraRed (AUR). We used the GPCR-APEX2/AUR assay to perform a genome-wide CRISPR interference screen focused on identifying genes regulating expression and trafficking of the δ-opioid receptor (DOR). We identified 492 genes consisting of both known and new regulators of DOR function. We demonstrate that one new regulator, DNAJC13, controls trafficking of multiple GPCRs, including DOR, through the endosomal-lysosomal pathway by regulating the composition of the endosomal proteome and endosomal homeostasis.

The Cutting-edge of CRISPR for Cancer Treatment and its Future Prospects.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a versatile technology that allows precise modification of genes. One of its most promising applications is in cancer treatment. By targeting and editing specific genes involved in cancer development and progression, CRISPR has the potential to become a powerful tool in the fight against cancer. This review aims to assess the recent progress in CRISPR technology for cancer research and to examine the obstacles and potential strategies to address them. The two most commonly used CRISPR systems for gene editing are CRISPR/Cas9 and CRISPR/Cas12a. CRISPR/Cas9 employs different repairing systems, including homologous recombination (HR) and nonhomologous end joining (NHEJ), to introduce precise modifications to the target genes. However, off-target effects and low editing efficiency are some of the main challenges associated with this technology. To overcome these issues, researchers are exploring new delivery methods and developing CRISPR/Cas systems with improved specificity. Moreover, there are ethical concerns surrounding using CRISPR in gene editing, including the potential for unintended consequences and the creation of genetically modified organisms. It is important to address these issues through rigorous testing and strict regulations. Despite these challenges, the potential benefits of CRISPR in cancer therapy cannot be overlooked. By introducing precise modifications to cancer cells, CRISPR could offer a targeted and effective treatment option for patients with different types of cancer. Further investigation and development of CRISPR technology are necessary to overcome the existing challenges and harness its full potential in cancer therapy.

Impact of predictive selection of LbCas12a CRISPR RNAs upon on- and off-target editing rates in soybean.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized creating targeted genetic variation in crops. Although CRISPR enzymes have been reported to have high sequence-specificity, careful design of the editing reagents can also reduce unintended edits at highly homologous sites. This work details the first large-scale study of the heritability of on-target edits and the rate of edits at off-target sites in soybean (Glycine max), assaying ~700 T1 plants each resulting from transformation with LbCas12a constructs containing CRISPR RNAs (crRNAs) predicted to be either "unique" with no off-target sites or "promiscuous" with >10 potential off-targets in the soybean genome. Around 80% of the on-target edits observed in T0 plants were inherited in the T1 generation, and ~49% of the total observed on-target edits in T1 were not observed at T0, indicating continued activity of LbCas12a throughout the life cycle of the plant. In planta editing at off-target sites was observed for the Promiscuous but not the Unique crRNA. Examination of the edited off-target sites revealed that LbCas12a was highly tolerant to mismatches between the crRNA and target site in bases 21-23 relative to the start of the protospacer, but even a single mismatch in the first 20 nt drastically reduced the editing rate. In addition, edits at off-target sites have lower inheritance rates than on-target edits, suggesting that they occur later in the plant's lifecycle. Plants with a desired on-target edit and no off-target edits could be identified in the T1 generation for 100% of the T0 plants edited with the Unique crRNA compared with the 65% of T0 plants edited with the Promiscuous crRNA. This confirms that proper crRNA selection can reduce or eliminate off-target editing. Even when potential off-target sites are predicted, plants containing only the intended edits can still be identified and propagated.

Therapeutic CRISPR epigenome editing of inflammatory receptors in the intervertebral disc

Low back pain (LBP) ranks among the leading causes of disability worldwide and generates a tremendous socioeconomic cost. Disc degeneration, a leading contributor to LBP, can be characterized by the breakdown of the extracellular matrix of the intervertebral disc (IVD), disc height loss, and inflammation. The inflammatory cytokine TNF-α has multiple signaling pathways, including proinflammatory signaling through Tumor Necrosis Factor Receptor 1 (TNFR1), and has been implicated as a primary mediator of disc degeneration. We tested our ability to regulate the TNFR1 signaling pathway in vivo, utilizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenome editing to slow the progression of disc degeneration in rats. Sprague-Dawley rats were treated with TNF-α and CRISPR interference (CRISPRi)-based epigenome-editing therapeutics targeting TNFR1, showing decreased behavioral pain in a disc degeneration model. Surprisingly, while treatment with the vectors alone was therapeutic, the TNF-α injection became therapeutic after TNFR1 modulation. These results suggest direct inflammatory receptor modulation as a potent strategy for treating disc degeneration.

IFIT3 mediates TBK1 phosphorylation to promote activation of pDCs and exacerbate systemic sclerosis in mice.

To assess the impact of the IFIT3/TBK1 signalling pathway in activating plasmacytoid dendritic cells (pDCs) and its role in the development of SSc. Utilized single-cell RNA sequencing (scRNA-seq) and high-throughput transcriptome RNA sequencing to reveal the differential abundance of pDCs and the role of the key gene IFIT3 in SSc. Conducted in vitro cell experiments to evaluate the effect of IFIT3/TBK1 signalling pathway intervention on pDC activation cytokine release and fibroblast function. Constructed an IFIT3-/- mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to assess the potential benefits of intervening in the IFIT3/TBK1 signalling pathway on skin and lung fibrosis in the SSc mouse model. The IFIT3/TBK1 signalling pathway plays a crucial role in activating pDCs, with IFIT3 acting as an upstream regulator of TBK1. Intervention in the IFIT3/TBK1 signalling pathway can inhibit pDC activation cytokine release and impact fibroblast function. The IFIT3-/- mouse model shows potential benefits of targeting the IFIT3/TBK1 signalling pathway in reducing skin and lung fibrosis in the SSc mouse model. This study provides new insights into potential therapeutic targets for SSc, highlighting the critical role of the IFIT3/TBK1 signalling pathway in SSc development. This study elucidates the pivotal role of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc). This study identified the key regulatory gene involved in systemic sclerosis (SSc) as IFIT3. This study has found that IFIT3 functions as an upstream regulatory factor, activating TBK1. This study provides Evidence of the regulatory effects of the IFIT3/TBK1 pathway on plasmacytoid dendritic cells (pDCs). This study validated the therapeutic potential using the IFIT3-/- mouse model.

CRISPR-CAS9-MEDIATED EX VIVO GENE EDITING FOR INHERITED HEMATOLOGICAL DISORDERS: ADVANCEMENTS, CHALLENGES, AND CLINICAL POTENTIAL

Global healthcare systems have a great challenge in the form of inherited hematological diseases, which necessitates the development of new remedial strategies. By precisely targeting inherited abnormalities, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated ex vivo gene editing has surfaced as a promising approach to treat these diseases. This review offers a comprehensive examination of the advancements, challenges, and clinical eventuality of CRISPR-Cas9-intermediated ex vivo gene editing for inherited hematological diseases. With advancements in CRISPR-Cas9 technology, the eventuality to correct inheritable mutations responsible for inherited hematological diseases is within reach. However, challenges such as off-target effects, immune responses, and ethical considerations need to be addressed for the safe and effective perpetration of this technology. A promising understanding of how CRISPR-Cas9-intermediated gene editing functions in practice is handed by ongoing clinical studies, giving rise to the possibility of advanced remedial approaches and bettered patient issues. By addressing these complications in a human-readable format, this review attempts to provide greater understanding and appreciation for the eventuality of CRISPR-Cas9 technology in revolutionizing the treatment landscape for these challenging disorders and contribute to the ongoing discussion in the field and facilitate further exploration towards effective treatments for these challenging disorders.