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Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System

Authors :
MINA KOLAHDOUZMOHAMMADI
SARA Pahlavan
YASER Tahamtani
Fattah Sotoodehnejadnematalahi
MEHDI Totonchi
Source :
مجله علمی دانشگاه علوم پزشکی کردستان, Vol 27, Iss 4, Pp 93-109 (2022)
Publication Year :
2022
Publisher :
Kurdistan University of Medical Sciences, 2022.

Abstract

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.

Details

Language :
Persian
ISSN :
1560652X and 23454040
Volume :
27
Issue :
4
Database :
Directory of Open Access Journals
Journal :
مجله علمی دانشگاه علوم پزشکی کردستان
Publication Type :
Academic Journal
Accession number :
edsdoj.8abe490458fd47e08c48a7f6e8f1a78a
Document Type :
article