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Review
Bacterial recombineering - genome engineering via phage-based homologous recombination Gur Pines, Emily F. Freed, James D. Winkler, and Ryan T. Gill ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00009 • Publication Date (Web): 09 Apr 2015 Downloaded from http://pubs.acs.org on April 14, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Bacterial recombineering: genome engineering via phage-based homologous recombination Gur Pines, Emily F. Freed, James D. Winkler, and Ryan T. Gill∗ Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA E-mail: [email protected]
Abstract The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.
Keywords recombineering, lambda phage, homologous recombination, genome engineering ∗
To whom correspondence should be addressed
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Introduction Recombineering, a term that describes recombination-based genome engineering methods, addresses a key limitation in the generation of precisely modified bacterial genomes (1 ). Specifically, recombineering methods harness phage and/or native recombination systems to enable highly efficient targeted DNA integration (2 ). While recombineering can include the use of helper organisms (such as yeast) to perform the desired homology-based recombination, in this review we choose to focus on those systems capable of supporting in situ (in organism) editing. The continued development of fast, accurate, and relatively low-cost DNA synthesis has further enabled recombineering based genome engineering methods (3 – 8 ). Genome engineering represents a new frontier for metabolic engineering and synthetic biology, enabling the large-scale modification of multiple genomic targets in a given microbe for chemical production, information processing, and other areas of interest (9 –11 ). Further tandem improvements in synthesis, recombination efficiency, and other allied technologies promise to increase the effectiveness of recombineering as a research tool. Decades of advances in genetic engineering tools have allowed researchers to make ever more precise edits to their targeted genomes. While traditional efforts to insert genetic material into a heterologous host relied on plasmid-based expression or the addition of large, flanking homology arms for recombination into the host genome, the discovery of phageoriginated proteins allowing site-directed recombination in E. coli has enabled higher precision editing than ever before. These recombineering-led improvements have increased the scale of feasible experiments, including modifying the expression level of every single gene in a genome within a population (3 ) or replacement of every TAG stop codon in the E. coli genome (12 , 13 ). Coupled with increasingly inexpensive DNA synthesis, researchers can now design, amplify, and combine DNA constructs in a record time frame. It is clear that further advances in recombineering will translate into a concomitant increase in the scale of feasible genome modifications. In light of the speed and complexity of these advances, this review aims to consolidate 2 ACS Paragon Plus Environment
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the historical context of recombineering developments, current applications, and mechanistic information to provide a single resource for researchers to improve their understanding of recombination-based genome engineering in E. coli. A timeline for important developments enhancing the utility of recombineering or key applications is first presented, followed in turn by a review of current research relying on recombineering approaches to enable genome-scale engineering. The underlying recombination mechanism in E. coli along with an examination of the concept of recombineering efficiency is subsequently elucidated. Finally, we speculate on future developments in this area that will further expand our ability to rapidly and precisely engineer microbes at the genome level.
Timeline Although genome engineering is popularly classified as a relatively new field, engineering organisms for human-desired traits is an ancient practice. Early humans used selective breeding of plants and animals to evolve phenotypes beneficial to humans. These efforts relied upon selecting from standing genetic variation in each given population for the bulk of human history. Genome engineering today is largely based on the same principles: genetic variation is introduced into an organism, followed by screening or selection for organisms that have the desired phenotype. However, researchers no longer have to wait for evolution or induced mutagenesis to produce variation, as recent advances in DNA synthesis and sequencing technology have enabled writing and reading of any DNA sequence at largescales and low-costs. Many of the basic techniques required for the rational engineering of genomes were developed in the 1960s and 1970s (Figure 1). These techniques include the ability to synthesize DNA with nucleotides in a specified order (14 , 15 ), the ability to generate recombinant DNA constructs by cloning (16 , 17 ), and the ability to sequence DNA (18 ). The ability to read and write DNA, however, is not sufficient for genome engineering; the engineered DNA
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must also be incorporated into cells so it can carry out its function. Research done in the early 1980s showed that two different recombinase proteins could be used to create deletions, insertions, or inversions in a site-specific manner in the E. coli chromosome by flanking the DNA to be edited with a recombinase recognition site (19 , 20 ). Although modifying DNA in a site-specific manner was a significant advance for genome engineering, the need for recognition sites flanking the DNA makes these techniques cumbersome and the resulting scars are problematic for editing across the entire genome. It was not until fifteen years later that methods were developed in E. coli to edit the chromosome without the need for recombinase recognition sites. Two similar methods, ET cloning (21 ) and Lambda-Red recombination (22 ), both use bacteriophage proteins to mediate site-specific genome editing via homologous recombination. Insertions, deletions, or point mutations can be introduced into any location within the genome using either method by transforming either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) (23 ) with homology to the E. coli genome. The initial work relying on this approach involved editing a single gene at a time and therefore could be quite labor intensive (24 ). However, the advent of high-throughput DNA synthesis (25 ) combined with multiplex recombineering (3 , 4 ) have made it possible to rapidly create and screen genome-wide libraries in E. coli by synthesizing the necessary constructs en masse, followed by any necessary library construction and transformation.
Current applications of recombineering for genome engineering The advent of efficient, practical recombineering has significantly improved the ability of bioengineers to rapidly alter organisms in ways that are not feasible using older strain engineering tools. By leveraging homologous recombination in various contexts, researchers can now easily construct and integrate complex constructs or libraries in a site-directed man4 ACS Paragon Plus Environment
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ACS Synthetic Biology
ner, in contrast to more traditional cloning and insertion approaches (26 ). Two particular areas of genome and biological engineering have undergone profound changes due to the capabilities of recombineering tools, namely the rapid generation of strain designs and the construction of defined genome-scale libraries. Combined with DNA synthesis and powerful selection systems, such as CRISPR (27 ), recombineering will remain an integral tool in the genome engineering toolbox for the foreseeable future. The ability of researchers to build complex microbial strains in the context of metabolic engineering has dramatically increased as a result of increasingly facile genome modification through recombineering, aided by phage proteins or extensive homology between recombined regions. Since recombineering can also be used to repair DSBs (double stranded DNA breaks) through the integration of transformed DNA fragments, methods for making targeted DSBs (28 ) have increasingly been coupled with recombineering to allow for scarless integration of arbitrary DNA cassettes. DSB may also be induced by CRISPR-Cas9 to cleave unmodified DNA following transformation with recombineering cassettes to eliminate wild-type background (29 , 30 ). If a selectable marker is included, a shotgun transformation of DNA can be used to rapidly modify a range of host traits (31 ). Although these approaches to recombineering rely on short homologies introduced by PCR or chemical synthesis, many bacteria will also integrate large fragments of DNA using their native recombination machinery (32 ). This ability is exploited by conjugation-based approaches for genome engineering, where significant portions of the E. coli genome are transferred in a short time frame to rapidly generate diverse libraries using native recombination machinery (12 , 33 , 34 ). Other non-in situ approaches involve the use of a helper organism (typically yeast) to recombine fragments together, followed by transformation into the final bacterial host (35 , 36 ) as reviewed extensively elsewhere. Additional developments in recombineering for strain (re)construction promise to significantly expedite genome engineering projects in the future. While engineering a small number of strains has become dramatically simpler due to recombineering, highly efficient DNA integration coupled with the affordability of DNA
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synthesis allows researchers to construct truly genome-wide libraries that can be used to investigate complex tolerance and production phenotypes. In contrast to randomly constructed libraries (e.g. transposon insertion), defined libraries can be constructed using recombineering to ensure that every mutation is introduced at a specific location with a known downstream effect. For example, the E. coli collection of non-essential gene deletions (24 ) relied on the now popular Datsenko-Wanner Lambda-Red-based recombineering system (37 ) to manually construct approximately 4000 knockout strains. In addition to allowing the rapid screening of knockout phenotypes, this and similar collections can serve as resources for strain construction using phage transduction. Beyond genetic deletions, the decreasing cost of DNA synthesis has allowed researchers to design novel DNA constructs for integration with recombineering (Figure 2); the trackable multiplex recombineering (TRMR) method is a demonstration of this synergistic combination, where uniquely barcoded dsDNA cassettes that encode constitutive promoters or eliminate translation through ribosome binding site disruption were simultaneously integrated upstream of every gene in the E. coli genome using a single transformation (3 ). Each transformant contained a single cassette, and the TRMR library has subsequently been successfully used to identify the genetic bases of several tolerance and production phenotypes (38 , 39 ). A revamped E. coli barcoded deletion library is also under development (Y. Otsuka and colleagues and Freed and colleagues, unpublished results), which will simplify efforts to track the lineage relative fitness under selection. Recombineering using more targeted libraries has also been explored, principally using the multiplex automated genome engineering (MAGE) system (4 ), as a means to optimize metabolite production. The MAGE approach relies on the iterative integration of ssDNA oligos throughout the genome in order to generate a genotypically diverse population; larger scale efforts incorporating many rounds of MAGE coupled with hierarchical strain assembly (13 ) have been used for removing the TAG stop codon from the E. coli genetic code in a true genome scale application. Recently, in vivo generation of ssDNA substrates via reverse-
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transcriptase, followed by Lambda-Red induced recombineering, has been used to create a rewritable memory system in E. coli cells (40 ). These novel experimental approaches all rely on highly efficient DNA integration via recombineering, and would not be possible with traditional techniques for genome engineering. Although the success of recombineering for genome engineering is indisputable, there are multiple areas where technological limitations prevent researchers from fully exploiting the promise of this technology. Despite the relative low cost of synthesizing short DNA segments, synthesizing large DNA cassettes for recombineering remains limiting for many laboratories, as the cost per bp remains high (≈$1/bp for sequence-verified constructs). Although chip-based synthesis can be used for smaller, diverse constructs at a lower cost (up to $0.01/bp) (41 ), edits that cannot be contained within a single cassette of up to 150 bp are more challenging (3 ). Once the DNA cassette to be integrated is in hand, recombineering platform strains with disrupted mismatch repair systems are typically used as targets due to their increased recombination efficiency (42 ) (elaborated on below). If the number of required recombineering cycles is large, as occurs when altering numerous physically unlinked sites in the genome, the final strain can acquire a significant mutational load that may impact downstream processes (12 , 13 ); avoiding this issue by improving the effectiveness of recombineering in wild-type strains is therefore a key interest for recombineers. The key requirement unifying these distinct experimental approaches is their collective reliance on efficient, site-directed integration of foreign DNA into the target genome. If a selectable marker is included in the recombineered DNA, editing efficiencies (fraction of correct transformants) are high enough to permit the screening of a small number of positive clones following transformation and integration. However, it is desirable to avoid the incorporation of these markers, as their expression represents a diversion of cellular resources from human-desired activities, and may facilitate the spread of antibiotic markers into the environment. Moreover, large DNA fragment delivery to the genome might have unexpected and sometimes deleterious results. Best practices for quantifying and improving the efficiency of
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recombineering beyond the current state of the art are therefore under active investigation.
The recombineering mechanism E. coli performs homologous recombination at extremely low rates, on the order of 10−6 for typical recombineering substrates used for genome engineering (
Review
Bacterial recombineering - genome engineering via phage-based homologous recombination Gur Pines, Emily F. Freed, James D. Winkler, and Ryan T. Gill ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00009 • Publication Date (Web): 09 Apr 2015 Downloaded from http://pubs.acs.org on April 14, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Bacterial recombineering: genome engineering via phage-based homologous recombination Gur Pines, Emily F. Freed, James D. Winkler, and Ryan T. Gill∗ Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA E-mail: [email protected]
Abstract The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.
Keywords recombineering, lambda phage, homologous recombination, genome engineering ∗
To whom correspondence should be addressed
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Introduction Recombineering, a term that describes recombination-based genome engineering methods, addresses a key limitation in the generation of precisely modified bacterial genomes (1 ). Specifically, recombineering methods harness phage and/or native recombination systems to enable highly efficient targeted DNA integration (2 ). While recombineering can include the use of helper organisms (such as yeast) to perform the desired homology-based recombination, in this review we choose to focus on those systems capable of supporting in situ (in organism) editing. The continued development of fast, accurate, and relatively low-cost DNA synthesis has further enabled recombineering based genome engineering methods (3 – 8 ). Genome engineering represents a new frontier for metabolic engineering and synthetic biology, enabling the large-scale modification of multiple genomic targets in a given microbe for chemical production, information processing, and other areas of interest (9 –11 ). Further tandem improvements in synthesis, recombination efficiency, and other allied technologies promise to increase the effectiveness of recombineering as a research tool. Decades of advances in genetic engineering tools have allowed researchers to make ever more precise edits to their targeted genomes. While traditional efforts to insert genetic material into a heterologous host relied on plasmid-based expression or the addition of large, flanking homology arms for recombination into the host genome, the discovery of phageoriginated proteins allowing site-directed recombination in E. coli has enabled higher precision editing than ever before. These recombineering-led improvements have increased the scale of feasible experiments, including modifying the expression level of every single gene in a genome within a population (3 ) or replacement of every TAG stop codon in the E. coli genome (12 , 13 ). Coupled with increasingly inexpensive DNA synthesis, researchers can now design, amplify, and combine DNA constructs in a record time frame. It is clear that further advances in recombineering will translate into a concomitant increase in the scale of feasible genome modifications. In light of the speed and complexity of these advances, this review aims to consolidate 2 ACS Paragon Plus Environment
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ACS Synthetic Biology
the historical context of recombineering developments, current applications, and mechanistic information to provide a single resource for researchers to improve their understanding of recombination-based genome engineering in E. coli. A timeline for important developments enhancing the utility of recombineering or key applications is first presented, followed in turn by a review of current research relying on recombineering approaches to enable genome-scale engineering. The underlying recombination mechanism in E. coli along with an examination of the concept of recombineering efficiency is subsequently elucidated. Finally, we speculate on future developments in this area that will further expand our ability to rapidly and precisely engineer microbes at the genome level.
Timeline Although genome engineering is popularly classified as a relatively new field, engineering organisms for human-desired traits is an ancient practice. Early humans used selective breeding of plants and animals to evolve phenotypes beneficial to humans. These efforts relied upon selecting from standing genetic variation in each given population for the bulk of human history. Genome engineering today is largely based on the same principles: genetic variation is introduced into an organism, followed by screening or selection for organisms that have the desired phenotype. However, researchers no longer have to wait for evolution or induced mutagenesis to produce variation, as recent advances in DNA synthesis and sequencing technology have enabled writing and reading of any DNA sequence at largescales and low-costs. Many of the basic techniques required for the rational engineering of genomes were developed in the 1960s and 1970s (Figure 1). These techniques include the ability to synthesize DNA with nucleotides in a specified order (14 , 15 ), the ability to generate recombinant DNA constructs by cloning (16 , 17 ), and the ability to sequence DNA (18 ). The ability to read and write DNA, however, is not sufficient for genome engineering; the engineered DNA
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must also be incorporated into cells so it can carry out its function. Research done in the early 1980s showed that two different recombinase proteins could be used to create deletions, insertions, or inversions in a site-specific manner in the E. coli chromosome by flanking the DNA to be edited with a recombinase recognition site (19 , 20 ). Although modifying DNA in a site-specific manner was a significant advance for genome engineering, the need for recognition sites flanking the DNA makes these techniques cumbersome and the resulting scars are problematic for editing across the entire genome. It was not until fifteen years later that methods were developed in E. coli to edit the chromosome without the need for recombinase recognition sites. Two similar methods, ET cloning (21 ) and Lambda-Red recombination (22 ), both use bacteriophage proteins to mediate site-specific genome editing via homologous recombination. Insertions, deletions, or point mutations can be introduced into any location within the genome using either method by transforming either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) (23 ) with homology to the E. coli genome. The initial work relying on this approach involved editing a single gene at a time and therefore could be quite labor intensive (24 ). However, the advent of high-throughput DNA synthesis (25 ) combined with multiplex recombineering (3 , 4 ) have made it possible to rapidly create and screen genome-wide libraries in E. coli by synthesizing the necessary constructs en masse, followed by any necessary library construction and transformation.
Current applications of recombineering for genome engineering The advent of efficient, practical recombineering has significantly improved the ability of bioengineers to rapidly alter organisms in ways that are not feasible using older strain engineering tools. By leveraging homologous recombination in various contexts, researchers can now easily construct and integrate complex constructs or libraries in a site-directed man4 ACS Paragon Plus Environment
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ACS Synthetic Biology
ner, in contrast to more traditional cloning and insertion approaches (26 ). Two particular areas of genome and biological engineering have undergone profound changes due to the capabilities of recombineering tools, namely the rapid generation of strain designs and the construction of defined genome-scale libraries. Combined with DNA synthesis and powerful selection systems, such as CRISPR (27 ), recombineering will remain an integral tool in the genome engineering toolbox for the foreseeable future. The ability of researchers to build complex microbial strains in the context of metabolic engineering has dramatically increased as a result of increasingly facile genome modification through recombineering, aided by phage proteins or extensive homology between recombined regions. Since recombineering can also be used to repair DSBs (double stranded DNA breaks) through the integration of transformed DNA fragments, methods for making targeted DSBs (28 ) have increasingly been coupled with recombineering to allow for scarless integration of arbitrary DNA cassettes. DSB may also be induced by CRISPR-Cas9 to cleave unmodified DNA following transformation with recombineering cassettes to eliminate wild-type background (29 , 30 ). If a selectable marker is included, a shotgun transformation of DNA can be used to rapidly modify a range of host traits (31 ). Although these approaches to recombineering rely on short homologies introduced by PCR or chemical synthesis, many bacteria will also integrate large fragments of DNA using their native recombination machinery (32 ). This ability is exploited by conjugation-based approaches for genome engineering, where significant portions of the E. coli genome are transferred in a short time frame to rapidly generate diverse libraries using native recombination machinery (12 , 33 , 34 ). Other non-in situ approaches involve the use of a helper organism (typically yeast) to recombine fragments together, followed by transformation into the final bacterial host (35 , 36 ) as reviewed extensively elsewhere. Additional developments in recombineering for strain (re)construction promise to significantly expedite genome engineering projects in the future. While engineering a small number of strains has become dramatically simpler due to recombineering, highly efficient DNA integration coupled with the affordability of DNA
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synthesis allows researchers to construct truly genome-wide libraries that can be used to investigate complex tolerance and production phenotypes. In contrast to randomly constructed libraries (e.g. transposon insertion), defined libraries can be constructed using recombineering to ensure that every mutation is introduced at a specific location with a known downstream effect. For example, the E. coli collection of non-essential gene deletions (24 ) relied on the now popular Datsenko-Wanner Lambda-Red-based recombineering system (37 ) to manually construct approximately 4000 knockout strains. In addition to allowing the rapid screening of knockout phenotypes, this and similar collections can serve as resources for strain construction using phage transduction. Beyond genetic deletions, the decreasing cost of DNA synthesis has allowed researchers to design novel DNA constructs for integration with recombineering (Figure 2); the trackable multiplex recombineering (TRMR) method is a demonstration of this synergistic combination, where uniquely barcoded dsDNA cassettes that encode constitutive promoters or eliminate translation through ribosome binding site disruption were simultaneously integrated upstream of every gene in the E. coli genome using a single transformation (3 ). Each transformant contained a single cassette, and the TRMR library has subsequently been successfully used to identify the genetic bases of several tolerance and production phenotypes (38 , 39 ). A revamped E. coli barcoded deletion library is also under development (Y. Otsuka and colleagues and Freed and colleagues, unpublished results), which will simplify efforts to track the lineage relative fitness under selection. Recombineering using more targeted libraries has also been explored, principally using the multiplex automated genome engineering (MAGE) system (4 ), as a means to optimize metabolite production. The MAGE approach relies on the iterative integration of ssDNA oligos throughout the genome in order to generate a genotypically diverse population; larger scale efforts incorporating many rounds of MAGE coupled with hierarchical strain assembly (13 ) have been used for removing the TAG stop codon from the E. coli genetic code in a true genome scale application. Recently, in vivo generation of ssDNA substrates via reverse-
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transcriptase, followed by Lambda-Red induced recombineering, has been used to create a rewritable memory system in E. coli cells (40 ). These novel experimental approaches all rely on highly efficient DNA integration via recombineering, and would not be possible with traditional techniques for genome engineering. Although the success of recombineering for genome engineering is indisputable, there are multiple areas where technological limitations prevent researchers from fully exploiting the promise of this technology. Despite the relative low cost of synthesizing short DNA segments, synthesizing large DNA cassettes for recombineering remains limiting for many laboratories, as the cost per bp remains high (≈$1/bp for sequence-verified constructs). Although chip-based synthesis can be used for smaller, diverse constructs at a lower cost (up to $0.01/bp) (41 ), edits that cannot be contained within a single cassette of up to 150 bp are more challenging (3 ). Once the DNA cassette to be integrated is in hand, recombineering platform strains with disrupted mismatch repair systems are typically used as targets due to their increased recombination efficiency (42 ) (elaborated on below). If the number of required recombineering cycles is large, as occurs when altering numerous physically unlinked sites in the genome, the final strain can acquire a significant mutational load that may impact downstream processes (12 , 13 ); avoiding this issue by improving the effectiveness of recombineering in wild-type strains is therefore a key interest for recombineers. The key requirement unifying these distinct experimental approaches is their collective reliance on efficient, site-directed integration of foreign DNA into the target genome. If a selectable marker is included in the recombineered DNA, editing efficiencies (fraction of correct transformants) are high enough to permit the screening of a small number of positive clones following transformation and integration. However, it is desirable to avoid the incorporation of these markers, as their expression represents a diversion of cellular resources from human-desired activities, and may facilitate the spread of antibiotic markers into the environment. Moreover, large DNA fragment delivery to the genome might have unexpected and sometimes deleterious results. Best practices for quantifying and improving the efficiency of
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recombineering beyond the current state of the art are therefore under active investigation.
The recombineering mechanism E. coli performs homologous recombination at extremely low rates, on the order of 10−6 for typical recombineering substrates used for genome engineering (
