The pCas/pTargetF system is one of the most popular genomic editing systems in E. coli. According to data from MolecularCloud, Addgene and our laboratory, there were 723 and 615 usage records in total for pCas and pTargetF respectively. However, few users pointed out that this system actually didn’t work in E. coli BL21(DE3) as transformants failed to grow up after the transformation of pTargetF into the BL21(DE3)/pCas strain. Sheng Yang Lab from Chinese Academy of Sciences optimized the pCas/pTargetF system and updated it into a pEcCas/pEcgRNA system. The work “A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli” is published in Acta Biochimica et Biophysica Sinica on 25 March，2021.
1. The pEcCas plasmid can be operated at 37℃, and the addition of a sacB gene renders it beneficial for plasmid clearance;
2. It is more convenient to update the N20 on pEcgRNA plasmid as only two 24nt oligos are required and the BsaI linearized pEcgRNA backbone can be prepared in large amounts and frozen for future use or directly ligated with the annealed 24nt oligos to generate new pEcgRNA;
3. pEcCas/pEcgRNA can be applied not only in E. coli K-12 strains, but also in E. coli B and W strains.
In this work, the pEcCas/pEcgRNA system was successfully tested in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain and 1 Tatumella citrea, with a total of 70 sites edited. Authors have also optimized the strategy for plasmid curing, providing users with a complete editing process. They hope to share the pEcCas/pEcgRNA to academia and industry to make up for the insufficient editing effect of pCas/pTargetF in E. coli and get feedbacks of pEcCas/pEcgRNA from the users.
All plasmids can be found on my personal page. Leave your comments if you have any questions about our research.