Application of CRISPR/Cas9 genome editing technology in hereditary retinal diseases / 中华实验眼科杂志

Several mutant genes for inherited retinal diseases have been identified, but effective treatments are still lacking.The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system can edit human genomic DNA by nonhomologous end joining or homology-directed repair, offering more possibilities for the treatment of hereditary retinal diseases.CRISPR/Cas9 not only can genetically correct patient-derived induced pluripotent stem cells (iPSCs) to observe their differentiation into retinal cells thereby, thereby exploring the pathogenesis of the disease and implementing cell therapy, but can also be delivered to the body via vectors and directly act on target cells to achieve in vivo gene editing.CRISPR/Cas9 gene editing technology in hereditary retinal diseases has been mainly used in retinitis pigmentosa, hereditary X-linked juvenile retinoschisis, and Leber congenital amaurosis 10, of which the in vitro application of CRISPR/Cas9 for Leber congenital amaurosis 10 has entered the clinical trial stage.In this paper, we reviewed the mechanism and key advances of CRISPR/Cas9 and provided an overview of gene editing in IRDs.

CRISPR/Cas9-mediated Knockout of KIFC1 Inhibits Proliferation and Induces Apoptosis of Cervical Cancer Cells / 肿瘤防治研究

Objective To investigate the functions of the KIFC1 gene in tumor cells and its effect on the proliferation of cervical cancer cells. Methods We designed sgRNAs targeting the KIFC1 gene and constructed a recombinant plasmid based on the pSpCas9 (BB)-2A-GFP vector, which was co-transfected into HeLa cells. We screened monoclonal knockout cell lines through flow cytometry sorting, limited dilution inoculation of cells, and sequencing. RT-qPCR, Western blot, and immunofluorescence were used to detect the transcription and protein expression levels of KIFC1 in knockout cells. Cell phenotypes such as nucleus and microtubule cytoskeleton were observed using phase-contrast microscopy and fluorescence confocal microscopy. Cell proliferation, cell cycle, and apoptosis were analyzed by growth curve plotting, EdU labeling, and acridine orange staining. Results The deletion of the KIFC1 gene resulted in the abnormal phenotypes of HeLa cells, with increased numbers of multinuclei, micronucleus, and disordered microtubules. The cell cycle was disrupted, accompanied with a significant increase in the ratio of late apoptotic cells and a decrease in cell proliferation (all P < 0.05). Conclusion KIFC1 gene deletion affects the assembly of microtubules and cell division in HeLa cells, leading to abnormal nuclear morphology, chromatin elimination, cell cycle arrest, and increased cell apoptosis.

Research progress on clustered regularly interspaced short palindromic repeats-associated 9 system for the treatment of HSV / 中华检验医学杂志

Herpes simplex virus (HSV) is a double-stranded DNA enveloped virus that causes severe effects on the human body by infecting the skin and nerve tissues. Because of latency and reactivation, the rapid detection and eradication of HSV are great challenges for clinical treatments. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has developed rapidly in the field of gene editing and detection due to its simple design and high targeting efficiency.

The progress of the gene editing therapy of inherited retinal diseases based on CRISPR/Cas9 / 中华眼底病杂志

Inherited retinal diseases (IRDs) are the major cause of refractory blinding eye diseases, and gene replacement therapy has already made preliminary progress in the treatment of IRDs. For IRDs that cannot be treated by gene replacement therapy, gene editing provides an alternative therapeutic method. Strategies like disruption of pathogenic variants with or without gene augmentation therapy and precise repair of pathogenic variants can be applied for IRDs with various inheritance patterns and pathogenic variants. In animal models of retinitis pigmentosa, Usher syndrome, Leber congenital amaurosis, cone rod cell dystrophy, and other disorders, CRISPR/Cas9, base editing, and prime editing showed the potential to edit pathogenic variations in vivo, indicating a promising future for gene editing therapy of IRDs.

Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness / 中国生物制品学杂志

@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.

CRISPR/Cas9-mediated endogenous TCR knockout enhances TCR-T cells targeted killing HPV16-positive cervical cancer cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：基于CRISPR/Cas9基因编辑技术制备无内源TCR的TCR-T细胞并鉴定其在体外杀伤HPV16阳性宫颈癌SiHa细胞的功能。方法：培养健康志愿者外周血CD8+ T细胞和Jurkat细胞，CRISPR/Cas9基因编辑技术敲除CD8+ T、Jurkat细胞的TCR基因，制备过表达转基因TCR的重组慢病毒，在敲除内源性TCR的CD8+ T和Jurkat细胞中用慢病毒过表达转基因TCR制备TCR-T细胞，多色FCM检测TCR-T细胞中TCR和CD3的表达水平，荧光素酶活性实验检测TCR-T细胞对HPV16阳性SiHa细胞的杀伤效率。结果：CRIPSR/Cas9基因编辑技术高效地敲除了外周血CD8+ T细胞和Jurkat细胞中的TRAC和TRBC基因，敲除效率分别为（81.4±4.5）%、（98.5±0.07）%，制备的无内源TCR的TCR-T细胞高效表达转基因TCR，在外周血CD8+ T和Jurkat细胞中表达率为（66.0±17.8）%、（97.3±2.6）%，敲除内源TRAC和TRBC基因有效增强CD8+ T和Jurkat细胞膜表达转基因TCR（均P<0.01），敲除内源TCR增强TCR-T细胞特异性杀伤HPV16阳性的SiHa细胞[（71.4±1.0）% vs （35.1±2.0）%，P<0.01）]。结论：无内源TCR的TCR-T细胞显著增强转基因TCR的表达和对HPV16阳性宫颈癌SiHa细胞的靶向杀伤能力，为提高TCR-T细胞的临床疗效提供了实验依据。

Research progress in the application of CRISPR/Cas9 technology in the treatment of cervical cancer / 国际生物医学工程杂志

Cervical cancer is the fourth-ranked malignant tumor of female cancer in the world, and it seriously threatens women’s health. The main treatment options for patients with cervical cancer are surgery or concurrent chemoradiotherapy. With the development of medical research, researchers are committed to exploring more effective and specific treatment options in order to increase the treatment options for cervical cancer and improve the treatment effect. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a method in which the Cas9 protein uses guide RNA (gRNA) to target the target gene and achieve precise editing of the target gene. At present, CRISPR/Cas9 technology has become a promising and powerful gene editing tool, a new and effective targeted therapy that has been applied in the treatment of various tumors. The research progress of CRISPR/Cas9 technology in the treatment of cervical cancer is mainly reviewed in terms of action targets, combination therapy strategies, and related drug resistance gene screening in order to provide new strategies for the treatment of cervical cancer.

High expression of MYH9 inhibits apoptosis of non-small cell lung cancer cells through activating the AKT/c-Myc pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).@*METHODS@#Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.@*RESULTS@#MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).@*CONCLUSION@#High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.

Application of CRISPR-Cas9 gene editing technology in crop breeding / 生物工程学报

The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.

Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans / 生物工程学报

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Development of porcine induced pluripotent stem cells with a CD163 reporter system / 生物工程学报

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Precision gene editing technologies based on CRISPR/Cas9: a review / 生物工程学报

Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Evidence of a synthetic lethality interaction between SETDB1 histone methyltransferase and CHD4 chromatin remodeling protein in a triple negative breast cancer cell line

During the tumorigenic process, cancer cells may become overly dependent on the activity of backup cellular pathways for their survival, representing vulnerabilities that could be exploited as therapeutic targets. Certain molecular vulnerabilities manifest as a synthetic lethality relationship, and the identification and characterization of new synthetic lethal interactions may pave the way for the development of new therapeutic approaches for human cancer. Our goal was to investigate a possible synthetic lethal interaction between a member of the Chromodomain Helicase DNA binding proteins family (CHD4) and a member of the histone methyltransferases family (SETDB1) in the molecular context of a cell line (Hs578T) representing the triple negative breast cancer (TNBC), a subtype of breast cancer lacking validated molecular targets for treatment. Therefore, we employed the CRISPR-Cas9 gene editing tool to individually or simultaneously introduce indels in the genomic loci corresponding to the catalytic domains of SETDB1 and CHD4 in the Hs578T cell line. Our main findings included: a) introduction of indels in exon 22 of SETDB1 sensitized Hs578T to the action of the genotoxic chemotherapy doxorubicin; b) by sequentially introducing indels in exon 22 of SETDB1 and exon 23 of CHD4 and tracking the percentage of the remaining wild-type sequences in the mixed cell populations generated, we obtained evidence of the existence of a synthetic lethality interaction between these genes. Considering the lack of molecular targets in TNBC, our findings provided valuable insights for development of new therapeutic approaches not only for TNBC but also for other cancer types.

CRISPR-Cas9 gene editing technology in human gene therapy: the new realm of medicine

Gene therapy has a huge clinical relevance in the present therapeutic world and is one of the many research fields of biology which received many benefits from the recent advancements of modern clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing technology. Researchers are on the way to make significant changes in the ways of treating genetic abnormalities. An increase in the number of approved clinical trials of CRISPR based gene therapy shows we are not too far from eliminating deadly diseases such as acquired immunodeficiency syndrome (AIDS), cancer and many inherited genetic conditions from the society. However, there are some challenges associated with the development of CRISPR technology in medical field most of which revolves around its safety, efficiency and ethics. Lack of an optimized method by which the CRISPR-Cas9 expression cassette can be delivered to cells is one of the main challenges when it comes to its application in human gene therapy. Although viral vectors are the most common delivery systems used in gene therapy, recent researches show promising results on using lipid-based l delivery systems such as liposome-templated hydrogel nanoparticles (LHNPs). As these could eliminate the safety concerns of using viral vectors, it is expected to have potential therapeutic applications in future. Nevertheless, the efficiency of non-viral systems is still not fully comparable with that of viral vectors. Hence, CRISPR based therapies might take longer than expected tobe prevalent in the medical field. In this short review, the recent advances of CRISPR technology in gene therapy is discussed along with its challenges and limitations.

Application of CRISPR-Cas9 to interrogate novel gene functions in cutaneous melanoma

Melanoma accounts for 3% of skin neoplasms and is the leading cause of death from skin disorders worldwide. The high mortality rate associated with this disease stems from the high capacity of melanoma patients to develop metastases and treatment relapse with inhibitors of the MAPK signaling pathway (such as BRAF inhibitors), commonly used in melanoma therapy. Thus, the investigation of genes involved in the mechanisms of melanoma development is essential for new and more effective therapeutic strategies. Hence, we describe in this thesis two projects involving the genes SIN3B and IRF4 as possible biomarkers for cutaneous melanoma. Initially, through bioinformatics analyses performed by our group, an upregulation of SIN3B was found in metastatic melanomas. This result together with the understanding of SIN3B role in regulating gene expression and oncogenic transformation, prompted us to describe in this thesis some mechanisms by which SIN3B may influence melanoma development. We then sought to characterize the gene function using SIN3B-deleted cells, generated by the CRISPR-Cas9 methodology. Initially, we observed increased SIN3B expression in BRAF-mutant metastatic melanomas, where we noted that the long splicing variant of the gene (NM_001297595.1) was effectively prevalent in melanomas. Subsequently, we designed gRNAs between the exons 2 and 3 of the human SIN3B gene and engineered three knockout clones and three control clones (containing empty lentiCRISPRv2 plasmid) from different melanoma cell lines (SKMEL28, A2058, and A375). Through functional analyses, it was observed that the absence of the gene did not interfere in the proliferation of tumor cells; however, it led to a decrease in invasive properties. These results were verified by Boyden chamber assays and transcriptome analysis (total RNA sequencing of deleted cells), where a decrease in migration and motility pathways was observed. Additionally, a screening of synthetically lethal genes with SIN3B was performed with a genome wide CRISPR library. These results showed that USP7 and STK11 genes, which belong to the FoxO signaling pathway, were essential in SIN3B-depleted melanoma cells. Finally, through a collaborative project with the Wellcome Trust Sanger Institute, previous large-scale sequencing analyses demonstrated that deletion of the IRF4 gene was lethal for melanoma cells. Accordingly, we performed IRF4 silencing in vitro and noticed that the lack of IRF4 promotes cell death and apoptosis, independently of MYC and MITF, known in the literature to be downstream targets of this gene. Therefore, these data suggest that IRF4 plays a vital role in melanoma cell survival. Taken together, both works herein described in this thesis demonstrate how CRISPR-Cas9 can be applied to study the functions and mechanisms of genes involved in melanoma progression, collectively helping in the development of more effective therapeutic strategies for this tumor

O melanoma representa 3% dos tipos de neoplasias cutâneas e é a maior causa das mortes por distúrbios de pele no mundo. A alta taxa de mortalidade associada à essa doença advém da alta capacidade de pacientes com melanoma desenvolverem metástases, e apresentarem recidiva após tratamento com inibidores da via de sinalização MAPK (como da proteína BRAF), comumente utilizados no tratamento de pacientes metastáticos. Assim, a investigação de genes envolvidos nos mecanismos de desenvolvimento do melanoma é primordial para novas estratégias terapêuticas mais efetivas. Dessa forma, descrevemos no presente trabalho dois projetos envolvendo os genes SIN3B e IRF4 como possíveis biomarcadores para melanoma cutâneo. Em análises prévias de bioinformática realizados pelo nosso grupo, SIN3B foi identificado tendo maior expressão em melanomas metastáticos. Além disso, diversos estudos mostraram que o gene está envolvido na regulação da expressão gênica e transformação oncogênica. Dessa forma, descrevemos nessa tese alguns mecanismos pelos quais SIN3B pode influenciar no desenvolvimento do melanoma, através da caracterização funcional de células SIN3B-deletadas pela metodologia CRISPR-Cas9. Inicialmente, observamos aumento na expressão de SIN3B em melanomas metastáticos BRAF-mutados, onde notamos que a variante de splicing longa do gene (NM_001297595.1), era efetivamente prevalente em melanomas. Assim, desenhamos sequências de RNA guias entre os éxons 2 e 3 do gene SIN3B humano e, obtivemos três clones knockout e outros três clones controle (contendo plasmídeo vazio) em diferentes linhagens de melanoma (SKMEL28, A2058 e A375), para caracterização funcional. Observou-se que a ausência do gene não interferiu na proliferação das células tumorais, contudo, acarretou na diminuição de processos invasivos. Esses resultados foram averiguados através de ensaios em câmara de Boyden e análises de transcriptoma (sequenciamento de RNA total das células deletadas), onde notou-se diminuição das vias de migração e motilidade. Adicionalmente, um rastreamento de genes sinteticamente letais com SIN3B foi realizado com uma biblioteca de CRISPR capaz de silenciar todo o genoma. Esses resultados mostraram que os genes USP7 e STK11, ambos pertencentes à via de sinalização de FoxO, são essenciais nas células SIN3B deletadas. Por fim, através de um projeto colaborativo com o Wellcome Trust Sanger Institute, análises prévias de sequenciamento de larga escala demonstraram que a deleção do gene IRF4 era letal para células de melanoma. Dessa forma, realizamos o silenciamento de IRF4 in vitro e notamos que a ausência do gene promove morte celular e apoptose, independentemente de MYC e MITF, conhecidos na literatura por serem alvos downstream do gene. Portanto, esses dados sugerem que IRF4 tem um papel importante na sobrevivência de células de melanoma. Em conjunto, ambos trabalhos descritos nessa tese, demonstram como a metodologia CRISPR-Cas9 pode auxiliar no entendimento de processos importantes para a malignidade do melanoma e contribuir para estratégias terapêuticas mais efetivas para esse tumor

Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein 9-mediated editing of Schistosoma mansoni genes: Identifying genes for immunologically potent drug and vaccine development

ABSTRACT Schistosomiasis is a neglected acute and chronic tropical disease caused by intestinal (Schistosoma mansoni and Schistosoma japonicum) and urogenital (Schistosoma haematobium) helminth parasites (blood flukes or digenetic trematodes). It afflicts over 250 million people worldwide, the majority of whom reside in impoverished tropical and subtropical regions in sub-Saharan Africa. Schistosomiasis is the second most common devastating parasitic disease in the world after malaria and causes over 200,000 deaths annually. Currently, there is no effective and approved vaccine available for human use, and treatment strongly relies on praziquantel drug therapy, which is ineffective in killing immature larval schistosomula stages and eggs already lodged in the tissues. The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated gene editing tool is used to deactivate a gene of interest to scrutinize its role in health and disease, and to identify genes for vaccine and drug targeting. The present review aims to summarize the major findings from the current literature reporting the usage of CRISPR/Cas9-mediated gene editing to inactivate genes in S. mansoni (acetylcholinesterase (AChE), T2 ribonuclease omega-1 (ω1), sulfotransferase oxamniquine resistance protein (SULT-OR), and α-N-acetylgalactosaminidase (SmNAGAL)), and freshwater gastropod snails, Biomphalaria glabrata (allograft inflammatory factor (BgAIF)), an obligatory component of the life cycle of S. mansoni, to identify their roles in the pathogenesis of schistosomiasis, and to highlight the importance of such studies in identifying and developing drugs and vaccines with high therapeutic efficacy.

Stratifin em Tumores de Cabeça e Pescoço: Um alvo molecular identificado por Phage Display / Stratifin in head and neck tumors: a molecular target identified by Phage Display

Nos últimos anos, houve um aumento na frequência dos casos de tumores de cabeça e pescoço apesar da diminuição do consumo do tabaco e álcool, e isso tem sido atribuído, em parte, à infecção pelo Papilomavírus Humano HPV. Por apresentar baixa sobrevida em 5 anos e ter alta morbidade, tem se buscado novos alvos moleculares para terapias combinadas. Nesse contexto nosso grupo identificou, através da tecnologia de Phage Display, uma sequência peptídica com interação preferencial por células tumorais com relação à células não transformadas, e ensaios adicionais identificaram seu alvo como sendo a proteína Stratifin. Stratifin tem sido reportado como um oncogene em diversos modelos tumorais, entretanto seu papel em carcinoma de células escamosas de cabeça e pescoço (CCECP) permanece desconhecido e poucos trabalhos na literatura reportam sua atividade em CCECP e/ou outro tumores relacionados ao HPV. Dessa forma, o objetivo desse trabalho foi explorar o potencial valor clínico e o papel biológico da Stratifin em CCECP. Dados do perfil de expressão e de metilação assim como dados clínicos foram extraídos em base de dados do The Cancer Genoma Atlas TCGA. Paralelamente, o perfil de expressão de Stratifin foi verificado através de ensaios de RT/qPCR e Western Blot em um painel de linhagens celulares de CCECP que contempla as principais características moleculares para esses tipos tumorais. A partir da observação de que todas as linhagens expressam Stratifin, utilizou-se a tecnologia de CRISPR/Cas9 para modular sua expressão (nocauteando ou superexpressando o gene) de modo a se observar parâmetros relacionados ao processo tumorigênico. Dessa forma, foi possivel verificar os efeitos da Stratifin em ensaios de proliferação, viabilidade após tratamentos com quimioterápicos, irradiação, crescimento livre de ancoragem e clonogenicidade. Como resultados, observamos que expressão aumentada de Stratifin no tecido tumoral quando comparado ao tecido normal, foi positivamente relacionada com o grau histológico, negatividade para HPV, mutação em TP53 e CDKN2A. Biologicamente, o nocaute de Stratifin foi relacionado com maior sensibilidade à quimioterápicos, menor capacidade de formação de colônias, e reduzida capacidade de crescimento livre de ancoragem. Esses resultados sugerem que Stratifin atue como um oncogene em CCECP, entretanto ensaios adicionais devem ser realizados para corroborar esse achados

Over recent years, there has been an increase of head and neck tumors frequency despite the decrease in tobacco and alcohol consumption, and this has been attributed, in part, to Human Papillomavirus infection. Due to its low 5-year survival and high morbidity, new molecular targets for combined therapies have been sought. In this context, our group identified, through Phage Display technology, a peptide sequence with preferential interaction by tumor cells in relation to non-transformed cells, and further assays identified its target as the Stratifin protein. Stratifin has been reported as an oncogene in several tumor models, however its role in head and neck squamous cell carcinoma (HNSCC) remains unknown and few works in the literature report its activity in HNSCC and/or other HPV-related tumors. Therefore, the aim of this study was to explore the potential clinical value and biological role of Stratifin in HNSCC. Expression profile data as well as clinical data were extracted from The Cancer Genome Atlas - TCGA database. In parallel, the expression profile of Stratifin was verified through RT/qPCR and Western Blot assays in a panel of HNSCC cell lines that address the main molecular characteristics for these tumor types. Since all cell lines express Stratifin, CRISPR/Cas9 technology was used to modulate its expression (gene knocking out or overexpressing) in order to check parameters related to the tumorigenic process. Thus, it was possible to verify the Stratifin effects in proliferation assays, viability after chemotherapy treatments, irradiation, anchorage-free growth and clonogenicity. As a result, we observed an increased expression of Stratifin in tumor tissue when compared to normal tissue, which was positively related to histological grade, HPV negativity, mutation in TP53 and CDKN2A. Biologically, knockout of Stratifin was associated with greater sensitivity to chemotherapy, less colony-forming capacity, and reduced anchorage-free growth capacity. These results suggest that Stratifin acts as an oncogene in HNSCC, however additional assays should be performed to corroborate these findings

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice / 亚洲男科学杂志(英文版)

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.