Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs

Background: the px330 and the related px459 plasmids are widely used for clusteredregularly interspaced short palindromic repeat (crispr)/cas9-mediated genomeediting. screening for plasmids containing the correct sgrna template insertionis one of the most important steps in this system. different methods for screening thesgrna inserts have been deployed. one such method is restriction enzyme (re)mapping. restriction enzyme mapping can be used to screen for numerous plasmidrecombinants simultaneously.methods: in this study, the sgrna templates were initially cloned into the abovepx459 plasmids. subsequently, the accuracy of the constructs was determined by remapping.results: this method was established to screen for sgrna-bearing px459 plasmids.however, numerous anomalies were detected after ligation of sgrna templates intore digested px459 plasmids.conclusion: our data suggest that re mapping is only appropriate as an initial screenand that the identity of all plasmids with the correctly identified re maps should beconfirmed by sanger sequencing.