RESUMEN
The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Herpesvirus Humano 1 , Evasión Inmune , Inmunidad Innata , Sistemas CRISPR-Cas/genética , Inmunidad Innata/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/genética , Evasión Inmune/genética , Humanos , Edición Génica/métodos , Animales , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Herpes Simple/inmunología , Herpes Simple/virología , Herpes Simple/genéticaRESUMEN
The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.
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Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Biblioteca de Genes , Inmunidad Innata , Inmunidad Innata/genética , Sistemas CRISPR-Cas/genética , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Línea Celular , Lentivirus/genéticaRESUMEN
Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.
Asunto(s)
Sistemas CRISPR-Cas , Mycoplasma pneumoniae , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/aislamiento & purificación , Humanos , Sistemas CRISPR-Cas/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Neumonía por Mycoplasma/diagnóstico , Neumonía por Mycoplasma/microbiologíaRESUMEN
Rationale: Regulatory processes of transcription factors (TFs) shape heart development and influence the adult heart's response to stress, contributing to cardiac disorders. Despite their significance, the precise mechanisms underpinning TF-mediated regulation remain elusive. Here, we identify that EBF1, as a TF, is highly expressed in human heart tissues. EBF1 is reported to be associated with human cardiovascular disease, but its roles are unclear in heart. In this study, we investigated EBF1 function in cardiac system. Methods: RNA-seq was utilized to profile EBF1 expression patterns. CRISPR/Cas9 was utilized to knock out EBF1 to investigate its effects. Human pluripotent stem cells (hPSCs) differentiated into cardiac lineages were used to mimic cardiac development. Cardiac function was evaluated on mouse model with Ebf1 knockout by using techniques such as echocardiography. RNA-seq was conducted to analyze transcriptional perturbations. ChIP-seq was employed to elucidate EBF1-bound genes and the underlying regulatory mechanisms. Results: EBF1 was expressed in some human and mouse cardiomyocyte. Knockout of EBF1 inhibited cardiac development. ChIP-seq indicated EBF1's binding on promoters of cardiogenic TFs pivotal to cardiac development, facilitating their transcriptional expression and promoting cardiac development. In mouse, Ebf1 depletion triggered transcriptional perturbations of genes, resulting in cardiac remodeling. Mechanistically, we found that EBF1 directly bound to upstream chromatin regions of cardiac hypertrophy-inducing genes, contributing to cardiac hypertrophy. Conclusions: We uncover the mechanisms underlying EBF1-mediated regulatory processes, shedding light on cardiac development, and the pathogenesis of cardiac remodeling. These findings emphasize EBF1's critical role in orchestrating diverse aspects of cardiac processes and provide a promising therapeutic intervention for cardiomyopathy.
Asunto(s)
Perfilación de la Expresión Génica , Miocitos Cardíacos , Transactivadores , Animales , Humanos , Ratones , Transactivadores/genética , Transactivadores/metabolismo , Miocitos Cardíacos/metabolismo , Diferenciación Celular/genética , Corazón/fisiopatología , Ratones Noqueados , Células Madre Pluripotentes/metabolismo , Transcriptoma/genética , Sistemas CRISPR-Cas/genéticaRESUMEN
The digestibility of starch is affected by amylose content, and increasing amylopectin chain length which can be manipulated by alterations to genes encoding starch-branching enzymes (SBEs). We investigated the impact of Cas9-mediated mutagenesis of SBEs in potato on starch structural properties and digestibility. Four potato starches with edited SBE genes were tested. One lacked SBE1 and SBE2, two lacked SBE2 and had reduced SBE1, and one had reduced SBE2 only. Starch structure and thermal properties were characterised by DSC and XRD. The impact of different thermal treatments on digestibility was studied using an in vitro digestion protocol. All native potato starches were resistant to digestion, and all gelatinised starches were highly digestible. SBE modified starches had higher gelatinisation temperatures than wild type potatoes and retrograded more rapidly. Gelatinisation and 18 h of retrogradation, increased gelatinisation enthalpy, but this did not translate to differences in digestion. Following 7 days of retrogradation, starch from three modified SBE starch lines was less digestible than starch from wild-type potatoes, likely due to the recrystallisation of the long amylopectin chains. Our results indicate that reductions in SBE in potato may be beneficial to health by increasing the amount of fibre reaching the colon after retrogradation.
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Enzima Ramificadora de 1,4-alfa-Glucano , Mutagénesis , Solanum tuberosum , Almidón , Solanum tuberosum/genética , Solanum tuberosum/química , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/química , Almidón/química , Almidón/metabolismo , Digestión , Sistemas CRISPR-Cas/genética , Amilopectina/química , Amilopectina/metabolismo , Amilosa/química , Amilosa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMEN
Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.
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Liofilización , Técnicas de Amplificación de Ácido Nucleico , Sistemas de Atención de Punto , Liofilización/métodos , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Sistemas CRISPR-Cas/genética , Indicadores y Reactivos/química , Técnicas de Diagnóstico Molecular/métodosRESUMEN
Sheath blight, caused by the fungus Rhizoctonia solani, is a major problem that significantly impacts rice production and can lead to substantial yield losses. The disease has become increasingly problematic in recent years due to the widespread use of high-yielding semi-dwarf rice cultivars, dense planting, and heavy application of nitrogenous fertilizers. The disease has become more challenging to manage due to its diverse host range and the lack of resistant cultivars. Despite utilizing traditional methods, the problem persists without a satisfactory solution. Therefore, modern approaches, including advanced breeding, transgenic methods, genome editing using CRISPR/Cas9 technology, and nanotechnological interventions, are being explored to develop rice plants resistant to sheath blight disease. This review primarily focuses on these recent advancements in combating the sheath blight disease.
Asunto(s)
Biotecnología , Sistemas CRISPR-Cas , Resistencia a la Enfermedad , Edición Génica , Oryza , Fitomejoramiento , Enfermedades de las Plantas , Rhizoctonia , Oryza/genética , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Resistencia a la Enfermedad/genética , Rhizoctonia/patogenicidad , Fitomejoramiento/métodos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Biotecnología/métodos , Plantas Modificadas Genéticamente/genética , Nanotecnología/métodosRESUMEN
CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2-12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r2 by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site's position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.
Asunto(s)
Sistemas CRISPR-Cas , Uso de Codones , Edición Génica , Uso de Codones/genética , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Biología Computacional/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Codón/genética , Expresión Génica/genéticaRESUMEN
An efficient electrochemiluminescence (ECL) emitter, Ir(ppy)3-based molecules has recently been reported to exhibit aggregation-induced electrochemiluminescence (AIECL) phenomenon. However, it remains a significant challenge to control the aggregation states of these molecules and achieve uniform aggregates with intense ECL emission. In this work, a biosensor was developed to detect microcystin-LR (MC-LR) based on Ir(ppy)3-functionalized zeolitic imidazolate framework-8 (Ir-ZIF-8) as the ECL emitter and the trans-cleavage activity of CRISPR-Cas12a as the methodological strategy. The Ir-ZIF-8, a functional metal-organic framework (MOF), exhibited the AIECL phenomenon via the spatial domain-limiting effect of encapsulating Ir(ppy)3 into the mesopores of ZIF-8, while the porosity and highly ordered topological structure of ZIF-8 effectively limited the molecular motion of Ir(ppy)3. CRISPR-Cas12a was employed to indiscriminately cleave double-stranded DNA decorated with carboxy tetramethylrhodamine (TAMRA), which quenched the ECL signal of Ir-ZIF-8 by resonance energy transfer and then separated the quencher from Ir-ZIF-8 to reactivate the signal. The concentration of MC-LR was designed to correlate with both the quencher amount and the activity of Cas12a. Then, two linear regression equations for MC-LR detection were constructed to improve the accuracy of the biosensor, and the constructed biosensor showed remarkable reproducibility, stability, and selectivity. The accurate detection of MC-LR with limits of detection of 1.2 and 5.9 pg/mL was made possible by the high quenching efficiency of TAMRA and the effective cutting ability of the editable CRISPR-Cas12a system.
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Técnicas Biosensibles , Sistemas CRISPR-Cas , Técnicas Electroquímicas , Mediciones Luminiscentes , Toxinas Marinas , Microcistinas , Microcistinas/análisis , Microcistinas/química , Toxinas Marinas/química , Sistemas CRISPR-Cas/genética , Técnicas Biosensibles/métodos , Zeolitas/química , Estructuras Metalorgánicas/química , Imidazoles/química , Límite de Detección , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/químicaRESUMEN
Herein, we report a target-triggered CRISPR/Cas12a assay by coupling lanthanide tagging and inductively coupled plasma mass spectrometry (ICP-MS) for highly sensitive elemental detection. Hepatitis B virus (HBV) DNA was chosen as a model analyte, and recombinase polymerase amplification (RPA) was used for target amplification. The double-stranded RPA amplicons containing a 5' TTTG PAM sequence can be recognized by Cas12a through a specific CRISPR RNA, activating the trans-cleavage activity of CRISPR/Cas12a and nonspecific cleavage of terbium (Tb)-ssDNA modified on magnetic beads (MBs). Following magnetic separation and acid digestion, the released Tb3+ ions were quantitated by ICP-MS and correlated to the concentration of HBV DNA. Taking advantage of the accelerated cleavage of Tb-ssDNA attached to the MB particles, RPA for target amplification, and ICP-MS for highly selective signal readout, this method permits the detection of 1 copy/µL of HBV DNA in serum with high specificity and holds great promise in the early diagnosis of viral infections or tumor development.
Asunto(s)
Sistemas CRISPR-Cas , ADN Viral , Virus de la Hepatitis B , Elementos de la Serie de los Lantanoides , Espectrometría de Masas , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , ADN Viral/genética , ADN Viral/análisis , Elementos de la Serie de los Lantanoides/química , Espectrometría de Masas/métodos , Sistemas CRISPR-Cas/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Recombinasas/metabolismoRESUMEN
In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.
Asunto(s)
Sistemas CRISPR-Cas , Dependovirus , Edición Génica , Técnicas de Inactivación de Genes , Factor de Transcripción STAT1 , Humanos , Dependovirus/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Células HEK293 , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Vectores Genéticos/genética , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Prokaryotic adaptive immune systems called CRISPR-Cas systems have transformed genome editing by allowing for precise genetic alterations through targeted DNA cleavage. This system comprises CRISPR-associated genes and repeat-spacer arrays, which generate RNA molecules that guide the cleavage of invading genetic material. CRISPR-Cas is classified into Class 1 (multi-subunit effectors) and Class 2 (single multi-domain effectors). Its applications span combating antimicrobial resistance (AMR), targeting antibiotic resistance genes (ARGs), resensitizing bacteria to antibiotics, and preventing horizontal gene transfer (HGT). CRISPR-Cas3, for example, effectively degrades plasmids carrying resistance genes, providing a precise method to disarm bacteria. In the context of ESKAPE pathogens, CRISPR technology can resensitize bacteria to antibiotics by targeting specific resistance genes. Furthermore, in tuberculosis (TB) research, CRISPR-based tools enhance diagnostic accuracy and facilitate precise genetic modifications for studying Mycobacterium tuberculosis. CRISPR-based diagnostics, leveraging Cas endonucleases' collateral cleavage activity, offer highly sensitive pathogen detection. These advancements underscore CRISPR's transformative potential in addressing AMR and enhancing infectious disease management.
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Infecciones Bacterianas , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Humanos , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/genética , Edición Génica , Bacterias/genéticaRESUMEN
Genome editing involves altering of the DNA in organisms including bacteria, plants, and animals using molecular scissors that helps in treatment and diagnosis of various diseases. Genome editing technology is exponentially growing and have been developed for enabling precise genomic alterations and the addition, removal, and correction of genes. These modifications begin with the creation of double-stranded breaks (DSBs) that is generated by nucleases and can be joined through homology-directed repair (HDR) or non-homologous end-joining (NHEJ). NHEJ is quick but increases mutation chances due to deletions and insertions of nucleotides at the break site, while HDR uses homologous templates for precise repair and targeted DNA specific to the gene or sequence. Other methods such as zinc-finger protein is a transcription factor that binds with DNA and binds specific to that sequence, which uniquely recognise 3-base pairs of DNA. TALENs consists of two domains: TALE domain, a transcription activator and FokI that is a restriction endonuclease that cuts the DNA at specific sites. CRISPR-Cas systems are clustered regularly interspersed short palindromic repeats present in various bacterial species. These sequences activate RNA-guided DNA cleavage, aiding in the development of an adaptive immune defence against foreign DNA. CRISPR-Cas9 is widely used for genome editing, regulation, diagnostic and many.
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Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Humanos , AnimalesRESUMEN
Cancer has been a primary contributor to morbidity and mortality worldwide. With an increasing trend of incidence and prevalence of cancer, progress has also been made in its treatment, starting from radiation and chemotherapy to immunotherapy and gene therapy. CRISPR-Cas technique, a promising gene editing tool, has been employed in cancer research for novel treatment regimens, identification of therapeutic targets, and unraveling the genetic mechanisms behind oncogenesis. CRISPR-based genome editing helped in identifying the roles of specific genetic factors linked to treatment resistance, metastasis, and cancer development. CRISPR allows the discovery of genes and treatment options through specifically interrupting tumor activators or activating tumor suppressor genes in cancer cells. Advancements in CRISPR technology, especially the use of immune cells like chimeric antigen receptor (CAR) T cells, has the potential to revolutionize personalized cancer treatment by precisely targeting and killing cancer cells. Furthermore, reactivating tumor suppressor genes makes cancer cells more susceptible to chemotherapy or immunotherapy. CRISPR-mediated genome editing can, hence, help to overcome resistance to traditional cancer treatments. The current manuscript covers that how is the CRISPR technology propelling revolutionary development in the field of cancer research, providing advance perspectives on the molecular causes of the disease and creating new lines for the development of more precise and potent cancer therapies.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Neoplasias , Humanos , Sistemas CRISPR-Cas/genética , Neoplasias/genética , Neoplasias/terapia , AnimalesRESUMEN
A body develops an autoimmune illness when its immune system mistakenly targets healthy cells and organs. Eight million people are affected by more than 80 autoimmune diseases. The public's and individuals' well-being is put at risk. Type 1 diabetes, lupus, rheumatoid arthritis, and multiple sclerosisare autoimmune diseases. Tissue injury, nociceptive responses, and persistent inflammation are the results of these stresses. Concerns about healthcare costs, health, and physical limitations contribute to these issues. Given their prevalence, it is crucial to enhance our knowledge, conduct thorough research, and provide all-encompassing support to women dealing with autoimmune diseases. This will lead to better public health and better patient outcomes. Most bacteria's immune systems employ CRISPR-Cas, a state-of-the-art technique for editing genes. For Cas to break DNA with pinpoint accuracy, a guide RNA employs a predetermined enzymatic pathway. Genetic modifications started. After it was developed, this method was subjected to much research on autoimmune diseases. By modifying immune pathways, CRISPR gene editing can alleviate symptoms, promote immune system tolerance, and decrease autoimmune reactivity. The autoimmune diseases that CRISPR-Cas9 targets now have no treatment or cure. Results from early clinical trials and preclinical studies of autoimmune medicines engineered using CRISPR showed promise. Modern treatments for rheumatoid arthritis,multiple sclerosis, and type 1 diabetes aim to alter specific genetic or immune mechanisms. Accurate CRISPR editing can fix autoimmune genetic disorders. Modifying effector cells with CRISPR can decrease autoimmune reactions. These cells include cytotoxic T and B lymphocytes. Because of improvements in delivery techniques and kits, CRISPR medications are now safer, more effective, and more accurately targeted. It all comes down to intricate immunological reactions and unexpected side consequences. Revolutionary cures for autoimmune problems and highly personalized medical therapies have been made possible by recent advancements in CRISPR.
Asunto(s)
Enfermedades Autoinmunes , Sistemas CRISPR-Cas , Humanos , Sistemas CRISPR-Cas/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/terapia , Enfermedades Autoinmunes/inmunología , Animales , Edición Génica , Terapia Genética/métodosRESUMEN
Protozoan parasitic diseases pose a substantial global health burden. Understanding the pathogenesis of these diseases is crucial for developing intervention strategies in the form of vaccine and drugs. Manipulating the parasite's genome is essential for gaining insights into its fundamental biology. Traditional genomic manipulation methods rely on stochastic homologous recombination events, which necessitates months of maintaining the cultured parasites under drug pressure to generate desired transgenics. The introduction of mega-nucleases (MNs), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) greatly reduced the time required for obtaining a desired modification. However, there is a complexity associated with the design of these nucleases. CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) is the latest gene editing tool that provides an efficient and convenient method for precise genomic manipulations in protozoan parasites. In this chapter, we have elaborated various strategies that have been adopted for the use of CRISPR-Cas9 system in Plasmodium, Leishmania and Trypanosoma. We have also discussed various applications of CRISPR-Cas9 pertaining to understanding of the parasite biology, development of drug resistance mechanism, gene drive and diagnosis of the infection.
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Sistemas CRISPR-Cas , Infecciones por Protozoos , Sistemas CRISPR-Cas/genética , Humanos , Infecciones por Protozoos/genética , Infecciones por Protozoos/parasitología , Animales , Edición GénicaRESUMEN
CRISPR-Cas systems have revolutionised precision medicine by enabling personalised treatments tailored to an individual's genetic profile. Various CRISPR technologies have been developed to target specific disease-causing genes in blood cancers, and some have advanced to clinical trials. Although some studies have explored the in vivo applications of CRISPR-Cas systems, several challenges continue to impede their widespread use. Furthermore, CRISPR-Cas technology has shown promise in improving the response of immunotherapies to blood cancers. The emergence of CAR-T cell therapy has shown considerable success in the targeting and correcting of disease-causing genes in blood cancers. Despite the promising potential of CRISPR-Cas in the treatment of blood cancers, issues related to safety, ethics, and regulatory approval remain significant hurdles. This comprehensive review highlights the transformative potential of CRISPR-Cas technology to revolutionise blood cancer therapy.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias Hematológicas , Humanos , Sistemas CRISPR-Cas/genética , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/genética , Animales , Edición Génica/métodosRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system possess a broad range of applications for genetic modification, diagnosis and treatment of infectious as well as non-infectious disease. The CRISPR-Cas system is found in bacteria and archaea that possess the Cas protein and guide RNA (gRNA). Cas9 and gRNA forms a complex to target and cleave the desired gene, providing defense against viral infections. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpesviruses, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cause major life threatening diseases which cannot cure completely by drugs. This chapter describes the present strategy of CRISPR-Cas systems for altering the genomes of viruses, mostly human ones, in order to control infections.
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Sistemas CRISPR-Cas , Edición Génica , Humanos , Sistemas CRISPR-Cas/genética , Virosis/genética , Virosis/terapia , Virosis/virología , Virus/genética , Genoma Viral/genéticaRESUMEN
The CRISPR-Cas9 method has revolutionized the gene editing. Epigenetic changes, including DNA methylation, RNA modification, and changes in histone proteins, have been intensively studied and found to play a key role in the pathogenesis of human diseases. CRISPR-While the utility of DNA and chromatin modifications, known as epigenetics, is well understood, the functional significance of various alterations of RNA nucleotides has recently gained attention. Recent advancements in improving CRISPR-based epigenetic modifications has resulted in the availability of a powerful source that can selectively modify DNA, allowing for the maintenance of epigenetic memory over several cell divisions. Accurate identification of DNA methylation at specific locations is crucial for the prompt detection of cancer and other diseases, as DNA methylation is strongly correlated to the onset as well as the advancement of such conditions. Genetic or epigenetic perturbations can disrupt the regulation of imprinted genes, resulting in the development of diseases. When histone code editors and DNA de-/ methyltransferases are coupled with catalytically inactive Cas9 (dCas9), and CRISPRa and CRISPRi, they demonstrate excellent efficacy in editing the epigenome of eukaryotic cells. Advancing and optimizing the extracellular delivery platform can, hence, further facilitate the manipulation of CRISPR-Cas9 gene editing technique in upcoming clinical studies. The current chapter focuses on how the CRISP/ Cas9 system provides an avenue for the epigenetic modifications and its employability for human benefit.
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Sistemas CRISPR-Cas , Epigénesis Genética , Humanos , Sistemas CRISPR-Cas/genética , Animales , Edición Génica/métodos , Metilación de ADN/genéticaRESUMEN
CRISPR-Cas technology has revolutionized microbiome research by enabling precise genetic manipulation of microbial communities. This review explores its diverse applications in gut microbiome studies, probiotic development, microbiome diagnostics, pathogen targeting, and microbial community engineering. Engineered bacteriophages and conjugative probiotics exemplify CRISPR-Cas's capability for targeted bacterial manipulation, offering promising strategies against antibiotic-resistant infections and other gut-related disorders. CRISPR-Cas systems also enhance probiotic efficacy by improving stress tolerance and colonization in the gastrointestinal tract. CRISPR-based techniques in diagnostics enable early intervention by enabling fast and sensitive pathogen identification. Furthermore, CRISPR-mediated gene editing allows tailored modification of microbial populations, mitigating risks associated with horizontal gene transfer and enhancing environmental and health outcomes. Despite its transformative potential, ethical and regulatory challenges loom large, demanding robust frameworks to guide its responsible application. This chapter highlights CRISPR-Cas's pivotal role in advancing microbiome research toward personalized medicine and microbial therapeutics while emphasizing the imperative of balanced ethical deliberations and comprehensive regulatory oversight.