Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs

Mehdi  Hassani; Sara   Hesami; Nahal  Maroofi; Mehdi  Banan

doi:10.18502/ajmb.v13i4.7211

Mehdi Hassani Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Sara Hesami Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Nahal Maroofi Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Mehdi Banan Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v13i4.7211

Keywords: CRISPR-Cas systems, Gene editing, Health services, Plasmids, Restriction mapping

Abstract

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.