Rapid Multicopy Chromosomal Integration Renovates the Way Cell Factories are Built

MulticopyChromosomal Integration Using CRISPR-Associated Transposases

Controlling the copy number of gene expression cassettes is an important strategy to engineer bacterial cells into high-efficiency biocatalysts. Current strategies mostly use plasmid vectors, but multicopy plasmids are often genetically unstable, and their copy numbers cannot be precisely controlled. The integration of expression cassettes into a bacterial chromosome has advantages, but iterative integration is laborious, and it is challenging to obtain a library with varied gene doses for phenotype characterization.

Here, we demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the Escherichia coli genome. Within 5 days without selection pressure, E. coli strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT E. coli containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to E. coli harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to Tatumella citrea further demonstrated that MUCICAT may be generally applied to many bacterial species.


Figure 1. Schematic illustration of multicopy chromosomal integration by CRISPR-associated transposases (MUCICAT).


All plasmids of this article can be found on my personal page. Leave your comments if you have any questions about our research.

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MolecularCloud July Newsletter: Multicopy Chromosomal Integration Using CRISPR-Associated Transposases


4 Reply

Dear Dr Yang, Congratulations on your fascinating work. I would like to understand what this multi copy loci originally encodes and what effect disruption of those genes has on the overall health and survival of those organisms. Also how many such multicopy loci are found on average?


Please refer to the references No.18-20 in "Multicopy Chromosomal Integration Using CRISPR-Associated Transposases".

Dear Dr. Sheng, You research encompanys many novel methods that has been used for E. coli however the results show that you have acheived a good copy number and the fold increase in productivity do supports that too. I have two querries regarding the methodology if you don't mind to assist me. Firstly, does this work can be done only on the industrial products can we utilize it for the production of vaccines against E. coli itself? Secondly, if possible can I have some ul of plasmids to work here in National University of Sciences and Technology (NUST), Islamabad, Pakistan?


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