RESUMEN
BACKGROUND Cancer is a life-threatening disease that affects approximately 18 million individuals worldwide. Breast cancer is the most common female neoplasm globally with more than 276,480 new cases of invasive breast cancer expected to be diagnosed in women in the U.S. alone in 2020. Genetic and epigenetic factors play role in the carcinogenesis and progression of this disease. In this study, MCF-7 adenocarcinoma cells were transfected with CRISPR/Cas9 plasmid to either knock out CDK11 or to activate CDH1. Treated cells were allografted into the mammary glands of female rats (150190 g, 68 weeks) to evaluate the capability of these cells to control cancer progression and metastasis. RESULTS qPCR data revealed a significant downregulation of CDK11 and upregulation of CDH1. Cell cycle analysis and apoptosis assays indicated the knockout of CDK11 and simultaneous activation of CDH1 resulted in cell cycle arrest at G2/M phase and accumulation of cells at G2. Meanwhile, the percentage of cells that underwent late apoptosis increased in both genome editing hits. Histopathological sectioning data indicated that untransfected MCF-7 cells were capable of developing tumors in the mammary gland and initiation g angiogenesis. Transfected cells significantly restricted cancer cell infiltration/invasion by minimally localizing tumors and inhibiting angiogenesis. CONCLUSIONS Although further investigation is needed, the present data indicate the potentiality of using CRISPR/Cas9-based therapy as a promising approach to treat breast cancer. Impact: these data indicate targeting cancer-related genes via any genome editing tool might represent a novel approach to combat cancer.
Asunto(s)
Animales , Femenino , Ratas , Neoplasias de la Mama/genética , Adenocarcinoma/genética , Proteínas Cdh1/genética , Proteína 9 Asociada a CRISPR/genética , Neoplasias de la Mama/secundario , Ratas Sprague-DawleyRESUMEN
Trichoderma harzianum is a filamentous fungus used as a biological control agent for agricultural pests. Genes of this microorganism have been studied, and their applications are patented for use in biofungicides and plant breeding strategies. Gene editing technologies would be of great importance for genetic characterization of this species, but have not yet been reported. This work describes mutants obtained with an auxotrophic marker in this species using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas (CRISPR-associated) system. For this, sequences for a guide RNA and Cas9 overexpression were inserted via biolistics, and the sequencing approach confirmed deletions and insertions at the pyr4 gene. Phenotypic characterization demonstrated a reduction in the growth of mutants in the absence of uridine, as well as resistance to 5-fluorotic acid. In addition, the gene disruption did not reduce mycoparasitc activity against phytopathogens. Thus, target disruption of the pyr4 gene in T. harzianum using the CRISPR/Cas9 system was demonstrated, and it was also shown that endogenous expression of the system did not interfere with the biological control activity of pathogens. This work is the first report of CRISPR Cas9-based editing in this biocontrol species, and the mutants expressing Cas9 have potential for the generation of useful technologies in agricultural biotechnology.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Hypocreales/genética , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genes FúngicosRESUMEN
Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06â¯mg/L (up to 4.8â¯mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.
Asunto(s)
Sistemas CRISPR-Cas , Ingeniería Metabólica/métodos , Yarrowia/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Medios de Cultivo/metabolismo , Fluorescencia , Proteínas Fúngicas/genética , Marcación de Gen , Mutación , ARN Guía de Kinetoplastida/genética , Uracilo/metabolismo , Yarrowia/crecimiento & desarrollo , Yarrowia/metabolismo , beta Caroteno/biosíntesis , beta Caroteno/genéticaRESUMEN
Easy, practical, and affordable gene silencing techniques are constantly progressing, and genetic tools such as TALEs, RNAi, and CRISPR/Cas9 have emerged as new techniques for understanding the basic biology and virulence mechanisms of pathogenic organisms, including bacteria. Here, we describe one-step targeted gene silencing in Leptospira biflexa by using plasmids expressing catalytically inactive Streptococcus pyogenes Cas9 (dCas9) and a single-guide RNA (sgRNA) capable of pairing to the coding strand of a desired gene.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Técnicas de Silenciamiento del Gen/métodos , Silenciador del Gen/fisiología , Leptospira/genética , ARN Catalítico/genética , ARN Guía de Kinetoplastida/genética , Sistemas CRISPR-Cas/genética , Plásmidos/genética , Streptococcus pyogenes/genéticaRESUMEN
While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells. In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.
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Proteína 9 Asociada a CRISPR/genética , Inactivación de Virus , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Sistemas CRISPR-Cas , Edición Génica , Regulación Viral de la Expresión Génica , Células HEK293 , VIH-1/genética , Humanos , ARN Guía de Kinetoplastida/genética , Replicación Viral/genéticaRESUMEN
The CRISPR-Cas9 system is a new tool that has been extensively used for genome editing. The system is composed of a Cas9 endonuclease, which has the function of cleaving DNA at a specific site, and a guide RNA (gRNA), which contains the sequence of the cleavage site that is the target of editing. Despite the great interest that has been generated because of the utility of Cas 9 as a molecular tool and a potential therapeutic protein, the production of the 158â¯kDa recombinant Cas9 protein derived from Streptococcus pyogenes remains a challenge. Here, we systematically evaluated the expression of recombinant Cas9 protein in two different E. coli strains in complex and defined media. The recombinant protein showed improved expression in E. coli BL21(DE3), while only traces of Cas9 protein could be detected in the Rosetta (DE3) strain as a result of much lower mRNA levels. The greatest Cas9 protein expression in defined media containing glucose was observed at an induction temperature of 30⯰C and with 8â¯h of post induction time using IPTG in shake flasks. The protein concentration obtained during a batch bioreactor culture was approximately 420.1â¯mg/L with 6â¯h of post induction time. The results demonstrated the possibility of efficient Cas9 protein expression in batch mode using E. coli BL21(DE3) and a simple defined medium and also showed the potential for further improvements that could facilitate large-scale production.
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Proteína 9 Asociada a CRISPR/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Proteína 9 Asociada a CRISPR/metabolismo , Medios de Cultivo/química , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Temperatura , Factores de TiempoRESUMEN
Drought episodes decrease plant growth and productivity, which in turn cause high economic losses. Plants naturally sense and respond to water stress by activating specific signalling pathways leading to physiological and developmental adaptations. Genetically engineering genes that belong to these pathways might improve the drought tolerance of plants. The abscisic acid (ABA)-responsive element binding protein 1/ABRE binding factor (AREB1/ABF2) is a key positive regulator of the drought stress response. We investigated whether the CRISPR activation (CRISPRa) system that targets AREB1 might contribute to improve drought stress tolerance in Arabidopsis. Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Stable transgenic plants expressing chimeric dCas9HAT were first generated. Then, we showed that the CRISPRa dCas9HAT mechanism increased the promoter activity controlling the ß-glucuronidase (GUS) reporter gene. To activate the endogenous promoter of AREB1, the CRISPRa dCas9HAT system was set up, and resultant plants showed a dwarf phenotype. Our qRT-PCR experiments indicated that both AREB1 and RD29A, a gene positively regulated by AREB1, exhibited higher gene expression than the control plants. The plants generated here showed higher chlorophyll content and faster stomatal aperture under water deficit, in addition to a better survival rate after drought stress. Altogether, we report that CRISPRa dCas9HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.
Asunto(s)
Aclimatación/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteína 9 Asociada a CRISPR/genética , Plantas Modificadas Genéticamente/fisiología , Factores de Transcripción/genética , Proteínas de Arabidopsis/metabolismo , Sistemas CRISPR-Cas/genética , Sequías , Regulación de la Expresión Génica de las Plantas/fisiología , Histona Acetiltransferasas , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/metabolismoRESUMEN
CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health-care system, and benefit-sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology.
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Anemia de Células Falciformes/prevención & control , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Edición Génica , Justicia Social , Anemia de Células Falciformes/terapia , Investigación Biomédica , Etnicidad , Terapia Genética , HumanosRESUMEN
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.