The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli
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Reisch_no-SCAR.pdf
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Author(s)
Reisch, Chris R.
Prather, Kristala L. Jones
Reisch, Christopher R.
Date Issued
October 2015
Journal
Scientific Reports
Publisher
Nature Publishing Group
Citation
Reisch, Chris R., and Kristala L. J. Prather. “The No-SCAR (Scarless Cas9 Assisted Recombineering) System for Genome Editing in Escherichia Coli.” Scientific Reports 5 (October 14, 2015): 15096.
Version
Final published version
Abstract
Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
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Creative Commons Attribution
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DOI of Published Version
https://doi.org/10.1038/srep15096
