Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs

Autor:	Sara Hesami, Nahal Maroofi, Mehdi Hassani, Mehdi Banan
Rok vydání:	2021
Předmět:	CRISPR-Cas systems Short Communication Biomedical Engineering Bioengineering Computational biology Gene editing Biology Restriction Enzyme Mapping Applied Microbiology and Biotechnology Health services Restriction mapping Plasmid Restriction map Genome editing CRISPR Plasmids Biotechnology
Zdroj:	Avicenna Journal of Medical Biotechnology
ISSN:	2008-4625 2008-2835
Popis:	Background: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. Methods: In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. Results: This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. Conclusion: Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9c2441ca26bd5fe1d9d9a78960f65933 https://doi.org/10.18502/ajmb.v13i4.7211 Zobrazit plný text záznamu