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Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System
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MINA KOLAHDOUZMOHAMMADI1    , SARA Pahlavan2 , YASER Tahamtani2 , Fattah Sotoodehnejadnematalahi3 , MEHDI Totonchi4 |
1- Ph.D. Candidate, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
2- Assistant Professor, Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Sq., Banihashem St., Resalat Highway, Tehran, Iran
3- Assistant Professor, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
4- Associate Professor, Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Banihashem Sq., Banihashem St., Resalat Highway, Tehran, Iran. , m.totonchi@royaninstitute.org |
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Abstract: (1691 Views) |
Background and Aim: Genome editing is an efficient and accurate method in biological and medical studies. Among the wide range of genome editing techniques, clustered regularly interspaced short palindromic repeats (CRISPR) is one of the simplest and yet promising methods. The CRISPR system consists of two key components; an endonuclease enzyme called Cas9 and guide RNA (gRNA), ensuring that Cas9 enzyme cuts at the right point in the genome. Although CRISPR genome editing is one of the useful methods to modify the genome, there are still multiple challenges in the technique steps.
Materials and Methods: sgRNAs were designed and ordered accordingly on the basis of the target site and vector of interest. Cloning procedures were performed and confirmed by sequencing as a gold standard. The list of high efficient sgRNAs for the LAMP-2 gene was prepared based on the available outstanding databases by analyzing and comparing the specificity and functionality as two main characteristics.
Results: In this study, we tried to evaluate the main challenges in the process of preparing CRISPR/Cas9 popular vectors (pX330/pX459). A list of high efficient sgRNAs for an autophagy gene is also prepared as an example for clarification of the sgRNA design procedures.
Conclusion: This study tried to depict problems that may be encountered during sgRNA design and plasmid preparation, followed by giving appropriate recommendations.
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| Keywords: CRISPR/Cas9, Genome editing, sgRNA, pX330/pX459, Plasmid |
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Full-Text [PDF 533 kb]
(790 Downloads)
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Type of Study: Original Research |
Subject:
Biotechnology
Received: 2021/11/7 | Accepted: 2021/12/6 | Published: 2022/12/14
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