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Multicopy Chromosomal Integration Using CRISPR-Associated Transposases
journal contribution
posted on 2020-07-03, 06:43 authored by Yiwen Zhang, Xiaoman Sun, Qingzhuo Wang, Jiaqi Xu, Feng Dong, Siqi Yang, Jiawei Yang, Zixu Zhang, Yuan Qian, Jun Chen, Jiao Zhang, Yingmiao Liu, Rongsheng Tao, Yu Jiang, Junjie Yang, Sheng YangControlling 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.
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Multicopy Chromosomal Integrationgenomic multicopy glucose dehydroge...gene expression cassettesmulticopy chromosomal integrationtarget multicopy lociuse plasmid vectors. coliRecombinant MUCICAT E . colistrategyEscherichia coli genomecrRNAprotein-expressing plasmid pET 24aE . coli strainsCRISPR-Associated Transposases Cont...copy numbers
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