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Presenter: Hassan, Argani, Tehran, Iran (Islamic Republic of)
STEM CELLS AND GENOME ENGINEERING
Hassan Argani 02Transplant Division, Shahidbeheshti university of medical sciences, Tehran, Iran (Islamic Republic of)
Pluripotent Stem Cells (PSC) are focused in the human genomic research. Manipulation of human genome produced opportunities for evaluating of normal and diseased genes, drug efficacy, innovation of gene-therapies, and performing site-specific manipulations within the genomes. Genome engineering could be used by Inducible Pluripotent Stem Cells (iPSC) which could develop into all three germ layers and is identified by absence of any chromosomal abnormalities, the detection of methylation patterns of iPSC, and finding of pluripotency markers. Editing of genomes is started with breaking in chromosomal DNA. Recently Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, derived from bacterial improved the genome editing, and caused widespread application of genome engineering techniques. CRISPR–Cas9 system is a potent system for genome editing. The most common types of genome manipulations performed using CRISPR–Cas9 include gene knockouts, or knock-ins through substitution of a target genetic sequence with a desired donor sequence. Two types of genome engineering will be happened: homologous or non-homologous DNA repair by the Cas9 nuclease. The delivery of the CRISPR–Cas9 and target donor vectors in hPSCs can be accomplished in multiple ways, including viral delivery and non-viral methods. The non-viral delivery methods includes lipid-mediated transfection (lipofection) and electroporation. It has become the most common and efficient in vitro delivery methods in hPSCs.The CRISPR–Cas9 system can be combined with iPSCs to generate single or multiple gene knock-outs, correct mutations, or insert reporter transgenes. Additionally knock-outs can also be utilized to investigate epigenetic roles and targets, such as investigating DNA methylation. CRISPR could be combined with hPSCs to explore the genetic determinants of lineage choice, differentiation, and stem cell fate, providing the ability to evaluate the contributions of various genes or noncoding elements to specific processes and pathways. CRISPR–Cas9 system plays also as a null or ‘nuclease-dead’ Cas9 (dCas9), which has no enzymatic activity but has been utilized through fusion with other functional protein domains. In conclusion RNA-guided genome targeting will have broad implications for synthetic biology, direct perturbation of gene networks, and targeted ex vivo and in vivo gene therapy in the near future.
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