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1.
CRISPR J ; 7(2): 73-87, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38635328

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing is evolving into an essential tool in the field of biological and medical research. Notably, the development of catalytically deactivated Cas9 (dCas9) enzyme has substantially broadened its traditional boundaries in gene editing or perturbation. The conjugation of dCas9 with various molecular effectors allows precise control over transcriptional processes, epigenetic modifications, visualization of chromosomal dynamics, and several other applications. This expanded repertoire of CRISPR-Cas9 applications has emerged as an invaluable molecular tool kit that empowers researchers to comprehensively interrogate and gain insights into health and diseases. This review delves into the advancements in Cas9 protein engineering, specifically on the generation of various dCas9 tools that have significantly enhanced the CRISPR-based technology capability and versatility. We subsequently discuss the multifaceted applications of dCas9, especially in interrogating the regulation and function of genes that involve in supporting cancer pathogenesis. In addition, we also delineate the designing and utilization of dCas9-based tools as well as highlighting its current constraints and transformative potentials in cancer research.


Assuntos
Edição de Genes , Neoplasias , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Epigênese Genética , Neoplasias/genética
2.
CRISPR J ; 6(3): 196-215, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37219623

RESUMO

Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic disorders. Precise base edits can be achieved using a variety of CRISPR-centric approaches, particularly homology-directed repair and the recently developed base editors and prime editors. Despite its much-touted potential, editing single DNA bases is technically challenging. In this review, we discuss the strategies for achieving exact base edits in the creation of various stem cell-based models for use in elucidating disease mechanisms and assessing drug efficacy, and the unique characteristics of stem cells that warrant special considerations.


Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas , Humanos , Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/metabolismo
3.
Int J Mol Sci ; 24(7)2023 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-37047421

RESUMO

Clear cell renal cell carcinoma (ccRCC) is a hypervascular tumor that is characterized by bi-allelic inactivation of the VHL tumor suppressor gene and mTOR signalling pathway hyperactivation. The pro-angiogenic factor PDGFB, a transcriptional target of super enhancer-driven KLF6, can activate the mTORC1 signalling pathway in ccRCC. However, the detailed mechanisms of PDGFB-mediated mTORC1 activation in ccRCC have remained elusive. Here, we investigated whether ccRCC cells are able to secrete PDGFB into the extracellular milieu and stimulate mTORC1 signalling activity. We found that ccRCC cells secreted PDGFB extracellularly, and by utilizing KLF6- and PDGFB-engineered ccRCC cells, we showed that the level of PDGFB secretion was positively correlated with the expression of intracellular KLF6 and PDGFB. Moreover, the reintroduction of either KLF6 or PDGFB was able to sustain mTORC1 signalling activity in KLF6-targeted ccRCC cells. We further demonstrated that conditioned media of PDGFB-overexpressing ccRCC cells was able to re-activate mTORC1 activity in KLF6-targeted cells. In conclusion, cancer cell-derived PDGFB can mediate mTORC1 signalling pathway activation in ccRCC, further consolidating the link between the KLF6-PDGFB axis and the mTORC1 signalling pathway activity in ccRCC.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Linhagem Celular Tumoral , Becaplermina/metabolismo , Neoplasias Renais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Supressora de Tumor Von Hippel-Lindau/genética
4.
Cells ; 11(10)2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35626649

RESUMO

Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations' molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.


Assuntos
Neoplasias da Mama , Sistemas CRISPR-Cas , Neoplasias da Mama/genética , Sistemas CRISPR-Cas/genética , Feminino , Células HEK293 , Humanos , Mutação/genética , Proteína Supressora de Tumor p53/genética , Fluxo de Trabalho
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