Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System
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...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | fas |
| Published: |
Kurdistan University of Medical Sciences
2022-10-01
|
| Series: | مجله علمی دانشگاه علوم پزشکی کردستان |
| Subjects: | |
| Online Access: | http://sjku.muk.ac.ir/article-1-7066-en.html |
| _version_ | 1797958054859243520 |
|---|---|
| author | MINA KOLAHDOUZMOHAMMADI SARA Pahlavan YASER Tahamtani Fattah Sotoodehnejadnematalahi MEHDI Totonchi |
| author_facet | MINA KOLAHDOUZMOHAMMADI SARA Pahlavan YASER Tahamtani Fattah Sotoodehnejadnematalahi MEHDI Totonchi |
| author_sort | MINA KOLAHDOUZMOHAMMADI |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T00:13:23Z |
| format | Article |
| id | doaj.art-8abe490458fd47e08c48a7f6e8f1a78a |
| institution | Directory Open Access Journal |
| issn | 1560-652X 2345-4040 |
| language | fas |
| last_indexed | 2024-04-11T00:13:23Z |
| publishDate | 2022-10-01 |
| publisher | Kurdistan University of Medical Sciences |
| record_format | Article |
| series | مجله علمی دانشگاه علوم پزشکی کردستان |
| spelling | doaj.art-8abe490458fd47e08c48a7f6e8f1a78a2023-01-09T06:12:38ZfasKurdistan University of Medical Sciencesمجله علمی دانشگاه علوم پزشکی کردستان1560-652X2345-40402022-10-0127493109Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 SystemMINA KOLAHDOUZMOHAMMADI0SARA Pahlavan1YASER Tahamtani2Fattah Sotoodehnejadnematalahi3MEHDI Totonchi4 Ph.D. Candidate, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran 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 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 Assistant Professor, Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran 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. 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.http://sjku.muk.ac.ir/article-1-7066-en.htmlcrispr/cas9genome editingsgrnapx330/px459plasmid |
| spellingShingle | MINA KOLAHDOUZMOHAMMADI SARA Pahlavan YASER Tahamtani Fattah Sotoodehnejadnematalahi MEHDI Totonchi Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System مجله علمی دانشگاه علوم پزشکی کردستان crispr/cas9 genome editing sgrna px330/px459 plasmid |
| title | Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System |
| title_full | Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System |
| title_fullStr | Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System |
| title_full_unstemmed | Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System |
| title_short | Step by Step Design of Popular and Common Vectors in Genetic Manipulation Using CRISPR/Cas9 System |
| title_sort | step by step design of popular and common vectors in genetic manipulation using crispr cas9 system |
| topic | crispr/cas9 genome editing sgrna px330/px459 plasmid |
| url | http://sjku.muk.ac.ir/article-1-7066-en.html |
| work_keys_str_mv | AT minakolahdouzmohammadi stepbystepdesignofpopularandcommonvectorsingeneticmanipulationusingcrisprcas9system AT sarapahlavan stepbystepdesignofpopularandcommonvectorsingeneticmanipulationusingcrisprcas9system AT yasertahamtani stepbystepdesignofpopularandcommonvectorsingeneticmanipulationusingcrisprcas9system AT fattahsotoodehnejadnematalahi stepbystepdesignofpopularandcommonvectorsingeneticmanipulationusingcrisprcas9system AT mehditotonchi stepbystepdesignofpopularandcommonvectorsingeneticmanipulationusingcrisprcas9system |