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1.
Parasitol Res ; 123(4): 176, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38573530

RESUMO

Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10-1 copies/µL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.


Assuntos
Giardia lamblia , Giardíase , Humanos , Animais , Bovinos , Giardia lamblia/genética , Sistemas CRISPR-Cas , Giardíase/diagnóstico , Giardíase/veterinária , Giardia/genética , Bioensaio
2.
Cell Stem Cell ; 31(4): 435-436, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38579680

RESUMO

Lee et al.1 analyzed the impacts of lentiviral vector transduction and CRISPR-Cas9/homology-directed repair editing on hematopoietic stem and progenitor cell (HSPC) engraftment and clonal dynamics. The study suggests that relative to lentiviral-vector-mediated gene addition, homology-directed repair editing is inefficient in vivo and might impair the engraftment and differentiation of HSPCs.


Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Animais , Humanos , Células-Tronco Hematopoéticas/metabolismo , Modelos Animais , Sistemas CRISPR-Cas/genética
3.
Planta ; 259(5): 112, 2024 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-38581602

RESUMO

MAIN CONCLUSION: The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.


Assuntos
Sistemas CRISPR-Cas , Oryza , Oryza/genética , Resistência à Doença/genética , Odorantes , Temperatura , Melhoramento Vegetal
4.
Pestic Biochem Physiol ; 200: 105810, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38582582

RESUMO

Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.


Assuntos
Mariposas , Animais , Mariposas/genética , Melaninas/genética , Sistemas CRISPR-Cas , Larva/genética , Pigmentação/genética
5.
Pestic Biochem Physiol ; 200: 105839, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38582601

RESUMO

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.


Assuntos
Sistemas CRISPR-Cas , RNA de Cadeia Dupla , Animais , Interferência de RNA , Spodoptera/genética , Spodoptera/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Insetos/genética , Larva/genética , Larva/metabolismo
6.
Sci Rep ; 14(1): 8095, 2024 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-38582932

RESUMO

Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.


Assuntos
Neoplasias da Mama , Sistemas CRISPR-Cas , Humanos , Feminino , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias da Mama/genética , Edição de Genes/métodos
7.
Open Biol ; 14(4): 240007, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38565160

RESUMO

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.


Assuntos
Edição de Genes , MicroRNAs , Animais , Camundongos , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Mutagênese , MicroRNAs/genética
8.
Sci Rep ; 14(1): 7820, 2024 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-38570576

RESUMO

Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/µl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.


Assuntos
Anaplasma marginale , Anaplasmose , Doenças dos Bovinos , Doenças Transmitidas por Carrapatos , Bovinos , Animais , Anaplasma marginale/genética , Anaplasmose/diagnóstico , Anaplasmose/genética , Sistemas CRISPR-Cas , Doenças dos Bovinos/genética , Doenças Transmitidas por Carrapatos/genética
9.
Nat Commun ; 15(1): 2901, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38575571

RESUMO

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.


Assuntos
Infecções por Vírus Epstein-Barr , MicroRNAs , Animais , Humanos , RNA/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Sistemas CRISPR-Cas/genética , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Mamíferos/genética
10.
Wiley Interdiscip Rev RNA ; 15(2): e1844, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38576085

RESUMO

RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m6A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.


Assuntos
Sistemas CRISPR-Cas , RNA , RNA/genética , Edição de Genes , Oligonucleotídeos Antissenso , Interferência de RNA
11.
Eur Rev Med Pharmacol Sci ; 28(6): 2430-2463, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38567606

RESUMO

Human Immunodeficiency Virus (HIV) has continuously been the greatest epidemic for humanity over a period spanning almost five decades. With no specific cure or treatment available to date despite extensive research, the C-C Chemokine Receptor 5, Delta 32 (CCR5 Δ32) allele genetic point mutation plays an imperative role in the prevention of acquired immunodeficiency syndrome (AIDS). This comprehensive study aims to review the induction of the homozygous recessive deletion genotype using the Clustered Regularly Interspaced Short Palindromic Repeats, Cas 9 Enzyme (CRISPR-Cas9), and hematopoietic stem cell transplantation under positive selection pressure for active immunity in seropositive patients' populations as the phenotype. A methodology is proposed to trigger a significant increase in the expression of Delta 32 beneficial mutant alleles within controlled modern healthcare facilities utilizing totipotent stem cells through somatic gene therapy. It acts upon two dysfunctional CCR5 genes, translating mutant G protein-coupled co-receptors, whose primary function is similar to that of C-X-C Motif Chemokine receptor 4 (CXCR4), by blocking the entry of viral RNA into the CD4+ T helper lymphocytes, halting infection and seizing viral life cycle. This modification is endemic in Northern Europe, where it naturally pertains to the Caucasian descent population samples in the form of polymorphism, p (X=0.01), where X is the probability of frequency of complete immunity against HIV-1 in population samples. The epigenetics of the single nucleotide polymorphism (SNP) are analyzed as they play a significant role in immunity distribution. Furthermore, a comparative analysis within the ethical boundaries of CRISPR-Cas9 is conducted to discuss the practical aspects and challenges of the presented methodologies and treatment alternatives. Additionally, the study assembles all available data and summarizes preexisting research while providing a promising solution to this ethical dilemma. Finally, a methodology is devised to answer the question of whether the variant-specific epidemic of AIDS caused by HIV-1 can be cured via artificially inducing immunity by CRISPR-Cas9.


Assuntos
Síndrome de Imunodeficiência Adquirida , Infecções por HIV , HIV-1 , Humanos , HIV-1/genética , Síndrome de Imunodeficiência Adquirida/genética , Síndrome de Imunodeficiência Adquirida/terapia , Infecções por HIV/genética , Infecções por HIV/terapia , Sistemas CRISPR-Cas/genética , Receptores CCR5/genética , Receptores CCR5/metabolismo , Mutação , Terapia Genética , Polimorfismo de Nucleotídeo Único , Frequência do Gene
12.
J Vis Exp ; (205)2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38557598

RESUMO

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ratos , Animais , Edição de Genes/métodos , Dependovirus/genética , Eletroporação/métodos , Zigoto
13.
PLoS One ; 19(4): e0301169, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38557903

RESUMO

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.


Assuntos
Solanum lycopersicum , Solanum lycopersicum/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Melhoramento Vegetal , Desenvolvimento Embrionário , Regeneração , Edição de Genes , Plantas Geneticamente Modificadas/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta
14.
Antonie Van Leeuwenhoek ; 117(1): 63, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38561518

RESUMO

Phage resistance is crucial for lactic acid bacteria in the dairy industry. However, identifying all phages affecting these bacteria is challenging. CRISPR-Cas systems offer a resistance mechanism developed by bacteria and archaea against phages and plasmids. In this study, 11 S. thermophilus strains from traditional yogurts underwent analysis using next-generation sequencing (NGS) and bioinformatics tools. Initial characterization involved molecular ribotyping. Bioinformatics analysis of the NGS raw data revealed that all 11 strains possessed at least one CRISPR type. A total of 21 CRISPR loci were identified, belonging to CRISPR types II-A, II-C, and III-A, including 13 Type II-A, 1 Type III-C, and 7 Type III-A CRISPR types. By analyzing spacer sequences in S. thermophilus bacterial genomes and matching them with phage/plasmid genomes, notable strains emerged. SY9 showed prominence with 132 phage matches and 30 plasmid matches, followed by SY12 with 35 phage matches and 25 plasmid matches, and SY18 with 49 phage matches and 13 plasmid matches. These findings indicate the potential of S. thermophilus strains in phage/plasmid resistance for selecting starter cultures, ultimately improving the quality and quantity of dairy products. Nevertheless, further research is required to validate these results and explore the practical applications of this approach.


Assuntos
Bacteriófagos , Streptococcus thermophilus , Streptococcus thermophilus/genética , Sistemas CRISPR-Cas , Iogurte , Bacteriófagos/genética , Plasmídeos/genética
15.
Zoolog Sci ; 41(2): 159-166, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38587910

RESUMO

Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.


Assuntos
Anthocidaris , Animais , Anthocidaris/genética , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Ouriços-do-Mar/genética , Edição de Genes/métodos
16.
Funct Integr Genomics ; 24(2): 70, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38565780

RESUMO

Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.


Assuntos
Oryza , Tolerância ao Sal , Tolerância ao Sal/genética , Sistemas CRISPR-Cas , Oryza/metabolismo , Estresse Salino , Mutação
17.
J Immunother Cancer ; 12(4)2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580329

RESUMO

BACKGROUND: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft. METHODS: We produced CB-CD8+ T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCRα (TRAC) and TCRß (TRBC) chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts. RESULTS: The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR-/- WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR+/+ WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR-/- WT1-TCR CD8+ CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells. CONCLUSIONS: In summary, we show the feasibility of developing a potent CB-derived CD8+ T cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.


Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Criança , Linfócitos T CD8-Positivos , Sistemas CRISPR-Cas/genética , Sangue Fetal , Receptores de Antígenos de Linfócitos T/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Linhagem Celular Tumoral , Recidiva
19.
Cell Mol Biol Lett ; 29(1): 48, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38589794

RESUMO

Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos
20.
J Agric Food Chem ; 72(14): 8180-8188, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38556749

RESUMO

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Feminino , Mariposas/genética , Mariposas/metabolismo , Larva/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Longevidade , Sistemas CRISPR-Cas , Endotoxinas/genética , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Resistência a Inseticidas/genética
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