Escherichia coli vectors having stringently repressible replication origins allow a streamlining of Crispr/Cas9 gene editing

This study describes the construction of an expanded series of plasmid vectors having replication origins of p15a, RSF1030 or RSF1031 that like pAM34 have IPTG-dependent replication. Surprisingly, these plasmids can be cured in fewer generations than pAM34. Derivatives of pAM34 with alternative antibiotic selection markers were also constructed. The utility of these vectors is demonstrated in the construction of a CRISPR-Cas9 system consisting of an IPTG-dependent Cas9 plasmid and a curable guide RNA plasmid having a streptomycin counterselection marker. This system was successfully demonstrated by construction of point mutations, deletions and insertions in the E. coli genome with a very high efficiency and in a shorter timescale than extant methods. The plasmids themselves were readily cured either together or singly from the resultant strains with minimal effort.
Source: Plasmid - Category: Biotechnology Source Type: research