The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli
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...
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2015
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| Online Access: | http://hdl.handle.net/1721.1/100529 https://orcid.org/0000-0003-0437-3157 https://orcid.org/0000-0002-4908-3914 |
| _version_ | 1811070035030966272 |
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| author | Reisch, Chris R. Prather, Kristala L. Jones Reisch, Christopher R. |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Reisch, Chris R. Prather, Kristala L. Jones Reisch, Christopher R. |
| author_sort | Reisch, Chris R. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T08:21:29Z |
| format | Article |
| id | mit-1721.1/100529 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:21:29Z |
| publishDate | 2015 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1005292022-09-30T09:10:15Z The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli Reisch, Chris R. Prather, Kristala L. Jones Reisch, Christopher R. Massachusetts Institute of Technology. Department of Chemical Engineering Massachusetts Institute of Technology. Synthetic Biology Center Reisch, Christopher R. Prather, Kristala L. Jones 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. Shell Global Solutions (US) National Institute of Food and Agriculture (U.S.) (Postdoctoral Fellowship 2013-67012-21022) 2015-12-28T13:52:15Z 2015-12-28T13:52:15Z 2015-10 2015-06 Article http://purl.org/eprint/type/JournalArticle 2045-2322 http://hdl.handle.net/1721.1/100529 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. https://orcid.org/0000-0003-0437-3157 https://orcid.org/0000-0002-4908-3914 en_US http://dx.doi.org/10.1038/srep15096 Scientific Reports Creative Commons Attribution http://creativecommons.org/licenses/by/4.0/ application/pdf Nature Publishing Group Nature Publishing Group |
| spellingShingle | Reisch, Chris R. Prather, Kristala L. Jones Reisch, Christopher R. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli |
| title | The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli |
| title_full | The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli |
| title_fullStr | The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli |
| title_full_unstemmed | The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli |
| title_short | The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli |
| title_sort | no scar scarless cas9 assisted recombineering system for genome editing in escherichia coli |
| url | http://hdl.handle.net/1721.1/100529 https://orcid.org/0000-0003-0437-3157 https://orcid.org/0000-0002-4908-3914 |
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