A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering

A - Papers appearing in refereed journals

Yuzbashev, T., Yuzbasheva, E. Y., Melkina, O. E., Patel, D., Bubnov, D., Dietz, H. and Ledesma-Amaro, R. 2023. A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering. Communications Biology. 6, p. 858. https://doi.org/10.1038/s42003-023-05202-5

AuthorsYuzbashev, T., Yuzbasheva, E. Y., Melkina, O. E., Patel, D., Bubnov, D., Dietz, H. and Ledesma-Amaro, R.
Abstract

CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid.

Year of Publication2023
JournalCommunications Biology
Journal citation6, p. 858
Digital Object Identifier (DOI)https://doi.org/10.1038/s42003-023-05202-5
Web address (URL)https://www.nature.com/articles/s42003-023-05202-5
Open accessPublished as ‘gold’ (paid) open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeBB/R01602X/1
Publisher's version
Output statusPublished
Publication dates
Online18 Aug 2023
Publication process dates
Accepted01 Aug 2023
PublisherNature Publishing Group
ISSN2399-3642

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