Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing

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Biotechnol. J. 2013, 8 DOI 10.1002/biot.201200237 www.biotechnology-journal.com

1 Introduction

Recombination has been an essential mechanism forgenetic engineering, particularly in microorganisms.Unfortunately, many organisms, such as the prototypicalbacterium, Escherichia coli, do not efficiently performrecombination with externally introduced DNA. As aresult, traditional genetic engineering in E. coli reliedheavily on phage-based transduction and conjugation inaddition to plasmid-based gene introduction. Recombi-neering, which combines the ease of electroporation andincreased recombination efficiencies of phage-based sys-tems, has revolutionized the speed and ease of genetic

engineering in E. coli. This technology has fundamental-ly changed the way we now think about editing genomesand has enabled large-scale genetic engineeringapproaches that were not possible previously. Specifical-ly, this technique takes advantage of heterologous expres-sion of bacteriophage proteins (Rec E/T from Racprophage [1, 2] or the Red αβγ proteins from Lambdaphage [3, 4]). When overexpressed in E. coli, these pro-teins enable efficient homologous recombination withDNA supplied by transformation. Any linear DNA frag-ment, either double (ds) or single stranded (ss), can bedesigned with an appropriate homology sequence (asshort as 20 base pairs (bp)) to introduce a variety ofchanges, such as point mutations, deletions, insertions,replacements, and inversions, into any DNA in vivo,including chromosomes [1, 3, 5], plasmids [1, 6], and bac-terial artificial chromosomes (BACs) [4, 7–9]. The firstlarge-scale use of recombineering in E. coli was the cre-ation of the Keio collection, which is a library of single-gene knockouts [10, 11]. Most recently, recombineeringhas been multiplexed and automated by using mixturesof ss- or ds-oligos to introduce a number of different

Review

Recombineering to homogeneity: extension of multiplexrecombineering to large-scale genome editing

Nanette R. Boyle1, T. Steele Reynolds1, Ron Evans2, Michael Lynch2 and Ryan T. Gill1

1 Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA2 OPX Biotechnologies, Inc, Boulder, CO, USA

Recombineering has been an essential tool for genetic engineering in microbes for many years andhas enabled faster, more efficient engineering than previous techniques. There have been numer-ous studies that focus on improving recombineering efficiency, which can be divided into threemain areas: (i) optimizing the oligo used for recombineering to enhance replication fork anneal-ing and limit proofreading; (ii) mechanisms to modify the replisome itself, enabling an increasedrate of annealing; and (iii) multiplexing recombineering targets and automation. These effortshave increased the efficiency of recombineering several hundred-fold. One area that has receivedfar less attention is the problem of multiple chromosomes, which effectively decrease efficiencyon a chromosomal basis, resulting in more sectored colonies, which require longer outgrowth toobtain clonal populations. Herein, we describe the problem of multiple chromosomes, discuss cal-culations predicting how many generations are needed to obtain a pure colony, and how changesin experimental procedure or genetic background can minimize the effect of multiple chromo-somes.

Keywords: Genome engineering · Lambda Red · Multiple chromosomes · Rec E/T · Recombineering efficiency

Correspondence: Prof. Ryan T. Gill, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, CO 80303, USA E-mail: [email protected]

Abbreviations: BAC, bacterial artificial chromosome; bp, base pairs; ds, dou-ble stranded; IPTG, β-D-1-thiogalactopyranoside; MAGE, multiplex auto-mated genome engineering; MMR, mismatch repair; PCR, polymerasechain reaction; ss, single stranded

Received 24 OCT 2012Revised 17 DEC 2012Accepted 22 JAN 2013

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2 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

changes simultaneously, thus greatly expanding our abil-ity to edit genomes on a larger scale [12–14]. Since thefirst report of phage-based recombineering in 1998 byStewart et al. [1] and Murphy [3], various efforts haveimproved our understanding of recombineering mecha-nisms and enabled further improvements in recombi-neering efficiencies. We present the current state ofknowledge on recombineering and then discuss key con-siderations and challenges to the extension of multiplexrecombineering to large-scale genome editing.

2 Bacteriophage recombineering in E. coli

Although homologous recombination has been reportedin E. coli, the number of reported recombinants was twoto three orders of magnitude lower (≈1 per 106 cells) thanthe native homologous recombination efficiencies of oth-er microbes, such as Saccharomyces cerevisiae. Howev-er, when bacteriophage-based recombination proteins,such as Rec E/T or Red αβγ, are expressed in E. coli,recombination efficiencies reach up to about 1 per 103–4

cells without any further optimization [1, 3, 15]. In the RecE/T system, RecE is an exonuclease with 5’ to 3’ activityand RecT is a ssDNA binding protein that stabilizes the ssDNA intermediate involved in annealing to the newlyintroduced complementary DNA strand during replica-tion [16, 17]. The Lambda Red system consists of threeproteins: Exo (also known as a), Beta, and Gam. Exo is anexonuclease, which degrades DNA in the 5’ to 3’ direction(similar to RecE); Beta is a ssDNA binding protein, whichbinds to ssDNA (similar to RecT), stabilizing it, and pro-tecting it from further action by exonucleases; and finallyGam inhibits RecBCD and SbcCD activity in the host,thereby protecting the exogenous DNA from beingdegraded by natural mechanisms [18, 19]. Comparing thetwo phage systems, RecE and Exo serve the same func-tion within the cell. The same is true for RecT and Beta,however, RecE does not function with Beta, and Exo doesnot function with RecT, so they are not interchangeable[20]. Recombineering with dsDNA requires the presenceof all three Lambda Red proteins, but ssDNA recombi-neering only requires the Beta protein [17, 21]. A recentstudy by Fu et al. [22] reported that the Rec E/T systemcould be used for direct DNA cloning; Rec E/T is highlyefficient at linear–linear homologous recombination andis more efficient than the Lambda Red proteins. Despitethis, the majority of current research efforts employ theLambda Red proteins, which is the main focus of the restof this review.

2.1 Current mechanism

Understanding of the mechanism of recombineering hasevolved significantly over the last few years. Initially, itwas thought that recombineering occurred by strand

invasion [23, 24]; however, in a recA– background, recom-bineering remains highly efficient and therefore a RecA–-independent mechanism was proposed, wherein thenewly introduced DNA is incorporated by annealing atreplication forks [23]. Stahl et al. [25] performed a detailedstudy to determine which mechanism was more likely,and recombination products from crosses of Lambdaphage showed characteristics more consistent withannealing. Later studies demonstrating enhanced target-ing of dsDNA recombineering to the lagging strand pro-vided further support for the replication-fork annealingmodel [26, 27]. The most recent mechanism (Fig. 1), pro-posed by Mosberg et al. [28], hypothesized that replica-tion-fork annealing occurred through a fully ss intermedi-ate. When dsDNA is used, the first step in recombinationis the degradation of one complete strand by Lambdaphage Exo. It is believed that Exo and Beta act synergis-tically, with Exo aiding in the binding of Beta to the ssintermediate [29]; thus overcoming any potential issueswith secondary structures of a fully ss intermediate. Betabinds to the ssDNA to protect it from further degradationand catalyzes its placement and annealing to the laggingstrand of the replication fork, acting as an Okazaki frag-ment [28, 30, 31]. The homology regions of the oligo bindto complementary regions and, during the next round ofreplication, the insertion (or deletion, mutation, etc.) isincorporated into the newly synthesized DNA. This pro-posed mechanism is supported by experimental evi-dence, such as higher efficiencies for oligos targeting thelagging strand [28, 32, 33] and only Beta is needed for ssDNA recombineering [17, 21]. Since the mechanism ofannealing to the chromosome is suspected to be the samefor both ss- and dsDNA, the main advantage of using dsDNA recombineering is that larger inserts can be cre-ated with the polymerase chain reaction (PCR) (possiblyincluding selectable markers) without any further pro-cessing to obtain ssDNA. This picture of the mechanismof recombineering is not complete, because recombina-tion does occur on the leading strand, but how that occursis not yet known.

2.2 Improvements in efficiency

With a better understanding of the recombineering mech-anism, there have been numerous recent studies focusedon improving the efficiency of Lambda Red based recom-bineering. In general, the most efficient methods nowentail (i) optimizing the oligo used for recombineering toenhance replication-fork annealing and limit proofread-ing; (ii) mechanisms to modify the replisome itself; and(iii) multiplexing recombineering targets and automation.

Sawitzke et al. [34] investigated the effect of increas-ing oligo concentration and found that saturationoccurred at 3000 oligos per cell. In the same study, theoptimal length was also investigated. The optimal lengthwas a 60- or 70-mer, but essentially oligos of 40–70 bases

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gave the same recombineering efficiencies, with recom-bineering efficiency decreasing rapidly for oligos of lessthan 25 nucleotides in length [34]. Wang et al. [13] inves-tigated the efficiency of recombineering with varyingsizes of insertions, deletions, and mismatches. For all cas-es, efficiency dropped off with increasing length of non-homologous sequence, such as mismatch, insertion, ordeletion; this indicates that minor rewriting can beachieved at much higher efficiencies than large changes.They also reported 90 bp as the optimal oligo length. Phos-phothionating the four nucleotides at the 5’ end of the oligo protects them from endogenous exonucleases andincreases efficiency two-fold [13]. Finally, designing oli-gos to target the lagging strand increases efficiency about30-fold [32, 33], which also supports the current mecha-nism of recombineering, which states that the oligo actsas an Okazaki fragment on the lagging strand of DNAreplication.

Several strategies for modifying the cellular machineryinvolved in DNA replication have been shown to be effec-tive, improving efficiency up to several hundred-fold. Thefirst major strategy was to inactivate the mismatch repair(MMR) system by knocking out MutS, which is responsi-

ble for correcting mistakes made during replication. Con-stantino and Court [32] reported that, by removing mutS,oligo-mediated recombination increased 400-fold for aG·G mismatch and 100-fold for most other mismatches.However, the basal rate of mutation increases 100-fold ina ΔmutS background, which can have undesired second-ary effects, in particular, when attempting to map engi-neered mutations onto selectable phenotypes. Oneapproach to circumvent the DNA MMR system is to usechemically modified bases that are not recognized by the MMR proteins. Wang et al. [35] used 2’-fluorouridine,5-methyl deoxycytidine, 2,6-diaminopurine, or isodeoxy -guanosine instead of the natural bases and found thatallelic replacement efficiencies increased 20-fold in astrain with 100-fold lower background mutation rate.Mutations were also introduced at higher efficiencies incells with a functional MMR system by introducing four ormore adjacent mismatches or introducing mismatches atfour or more consecutive wobble positions near the muta-tion site [34]. A more recent study introduced a knownmutation into the DNA primase to reduce the frequencyof priming for Okazaki fragments [36], which resulted inlonger Okazaki fragments and greater accessibility to

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Figure 1. Current proposed mechanism of Lambda Red recombineering in E. coli. Upon induction of Lambda Red proteins, the Gam protein blocks activityof RecBCD (ExoV) and SbcCD, which degrade exogenous DNA. The Exo protein degrades one DNA strand in the 5’ to 3’ direction and recruits Beta to bindthe exposed ssDNA to protect it from further degradation. Beta also promotes annealing to the lagging strand at the replication fork during DNA replica-tion. The ssDNA displaces an Okazaki fragment at the replication fork and becomes integrated into the newly synthesized DNA.



ssDNA on the lagging strand. This mutation, dnaGQ576A,resulted in 63% greater allelic replacement per clone thanthe wild type. A follow-up study removed five endogenousexonucleases (RecJ, ExoI, ExoVII, ExoX, and LambdaExo); these mutations alone increased recombineeringefficiency 46% and when combined with the dnaGQ576Amutation resulted in 111% more clones per cycle than thewild type; however, these mutations came at the expenseof cellular growth rate [36, 37].

Multiplexing and automation were also used toincrease the efficiency of recombineering. Wang et al. [13]built a device that automated all the steps in recombi-neering; thus enabling many more rounds of recombi-neering in one day. Multiplexing oligos also enabled theefficient creation of combinatorial libraries. This tech-nique, multiplex automated genome engineering(MAGE), was used to create combinatorial libraries con-taining about 3 billion mutants in a few days, several ofwhich were capable of producing five-fold more lycopene(mutations were directed at genes in the 1-deoxy-D-xylu-lose-5-phosphate (DXP) pathway) [13]. MAGE was alsoused to massively rewrite the E. coli chromosome, chang-ing all 314 TAG stop codons to TAA stop codons. In thisstudy, Isaacs et al. [12] speculated that there was a sub-population of cells that were highly recombinogenic, andthus, contained many more mutations than other cells.This discovery led to the development of the coselectionMAGE approach [38, 39]. The strategy here is that, sinceDNA is sufficiently unwound at the replication fork so thatup to 500 000 bp are exposed, then if one oligo is inte-grated into the chromosome, it is likely that a second (orthird, fourth, etc.) oligo designed to be integrated close bywould also be integrated. Therefore, coselection markers,which are easily screened or selected for (e.g. antibioticresistance or amino acid auxotrophy), are chosen aroundthe chromosome and oligos designed to turn them on/offare mixed in with the oligo mixture used during recombi-neering. The use of coselection greatly reduced the num-ber of colonies that had to be screened and resulted in theidentification of strains with as many as 12 mutations[39]. Sawitzke et al. [34] also reported increased recombi-neering efficiencies (≈100-fold) in cells co-electroporatedwith a selectable plasmid. As such, these techniques,along with access to cheap and accurate oligo libraries(up to several hundred thousand), have set the stage formassive genome engineering at a scale of dozens to hun-dreds of modifications in parallel and on laboratorytimescales.

3 Multiple chromosomes

It is widely known that E. coli can have multiple copies ofa chromosome within the cell, in fact, some reports havethe copy number of the chromosome as high as 16,depending on growth rate, growth stage, and nutrient


state in the medium [40, 41]. In batch culture, the numberof chromosomes within the cell varies widely (2 to 16)between stages of growth (lag, exponential, and station-ary) in rich medium and even in what is assumed to bebalanced, steady-state growth (exponential growth) thenumber of chromosomes per cell does not remain con-stant [42]. Variation in chromosome copy number is muchless in minimal medium, with cells having only betweenone to four copies [42, 43]; however, cells grown in mini-mal medium are less efficient at taking up exogenousDNA [28], and thus, reducing any potential advantage.Low efficiency recombineering in cells grown in minimalmedium can also be due to lower availability of replicationforks or other reasons yet to be elucidated. Another issueis that daughter cells inherit multiple chromosomes uponcell division as well, thus carrying on the mixed genotypethrough several generations.

3.1 Recombineering with multiple chromosomes

The presence of multiple genomes in the cell reduces theefficiency on a chromosomal basis significantly (Fig. 2).An ‘ideal’ cell with one chromosome per cell would resultin a maximum chromosomal efficiency of 50%, whereasthe presence of four chromosomes has the potential tobring that number down to 12.5%. If we define the effi-ciency in terms of strands of DNA instead of completechromosomes, then this decreases the efficiencies to 25%and 6.25%, respectively. Any cell with at least one chro-mosome containing the mutation would present theexpected phenotype; however, the degree of heterogene-ity within the population will be determined by the num-ber of chromosomes in the cell at the time of recombi-neering. During outgrowth, except in the ideal case,mutated cells would become diluted by wild type; anadditional complication arises when the mutation confersa growth defect, which would further dilute the desiredsubpopulation. Longer periods of outgrowth increase thelikelihood of finding a clonal colony; however, it alsoincreases the total number of cells, which need to bescreened/selected for to find the desired cells. This issueis briefly mentioned by Sawitzke et al. [34], who also pointto a figure of a plate with several sectored colonies platedonly 30 min after electroporation. Their recommendationis to allow the cells to grow for 3 h before plating to obtainpure colonies; a similar figure appears in Constantino andCourt [32]. While it is possible to identify a clonal colonyafter 3 h of outgrowth, longer outgrowth is needed toincrease the frequency of nonsectored colonies in theoverall population. If care is not taken to ensure mutantpopulations are homogeneous, mutants can be lost overtime if there is no selectable advantage.

To estimate the time required to obtain a homogenouspopulation, we constructed a simple mathematical mod-el. We have simplified the mechanism of recombineeringand replication by assuming one set of replication forks


per chromosome, chromosomes are split equally and ran-domly between daughter cells, and copy number is main-tained throughout the growth period. In the ideal case (1 chromosome per cell), only one generation is requiredto have nonsectored colonies. As one would suspect,increasing the chromosomal copy number results inlonger outgrowths to obtain chromosomal homogeneity. If

we assume a chromosomal recombineering efficiency of10%, it is estimated that 8 generations of outgrowth arerequired if 2 chromosomes are present per cell to obtain90% of recombinant phenotype cells with homogenouschromosomes. If 4 chromosomes are present per cell, 23 generations of outgrowth are required for the samedegree of recombinant homogeneity (approximately



Figure 2. The effect of multiple chromosomes on recombineering efficiency. (A) The ‘ideal’ case (one chromosome/cell) with a maximum theoretical effi-ciency of 50%. (B) A more typical case, in which the cell has multiple (4–8) chromosomes, which results in lower efficiencies, 12.5 to 50%. (C) The effect ofmultiple chromosomes on homogeneity of the population, assuming a recombineering efficiency of 10%, increasing the chromosome copy number from 1 to 8 requires 55 generations to obtain a clonal population (≈2 overnight plates).



1 overnight plate). If 8 chromosomes are present in eachcell, then 55 generations are required. An overnight plateis approximately 25 generations; therefore, cells with 4 or8 chromosomes require an extra 1 or 2 days, respectively,to obtain homogenous populations. These are crude esti-mates that ignore many of the more complicated detailsof recombineering and replication, which are discussed inmore detail below, but serve as a starting place for esti-mating how long one needs to wait to ensure a clonal pop-ulation.

This leads us to the question of how chromosomalcopy number can be controlled to increase the recombi-neering efficiency. As discussed previously, the type ofmedium used for growth can affect the chromosomal copynumber, but the trade-off is lower DNA uptake efficien-cies. In general, slower growth results in fewer chromo-somes (i.e. lower temperatures), but this increases theoverall time required for recombineering. Alternatively,one might attempt to engineer the intracellular mecha-nism that controls copy number. It is known that DnaAcontrols the initiation mass of DNA replication, and there-fore, controls copy number [44]. Temperature-sensitiveDnaA mutants (dnaA(Ts)) decrease replication initiationat higher temperatures and increase replication initiationat lower temperatures [45]. DnaA is autoregulated and theprotein concentration is rate-limiting for initiation[46–49]; Løbner-Olesen et al. [50] showed that, by placingdnaA expression under control of the lac promoter andoperator, the amount of DNA within the cell could be con-trolled by isopropyl β-D-1-thiogalactopyranoside (IPTG).Engineering replication machinery is a promising avenueto control copy number; further investigation into thisarea is likely to lead to the discovery of other methods toreduce DNA content in the cell and subsequentlyincrease recombineering efficiency, and thus, enable larg-er scale genome engineering.

3.3 Multiple replication forks

The simple example depicted in Fig. 2B does not give thewhole picture of how multiple genomes can affect recom-bineering. Replication of the chromosome requiresaround 40 min to complete, but cells are able to grow at amuch faster pace (td = 20 min). To maintain a high copynumber of the chromosome, the cell must initiate anoth-er round of replication before the previous one is com-plete. Therefore, at any given time, multiple replicationforks will be open simultaneously [51]. Since cells used forrecombineering are typically grown in LB medium andcollected and washed at the mid-exponential growthstage, they will almost assuredly have multiple replicationforks open. If the oligo that introduces the desired muta-tion is integrated into the chromosome at the leadingreplication fork (and onto the parent chromosome), thechromosomal efficiency will actually be enhancedbecause all subsequent DNA replication will incorporate

that mutation into the new chromosome. If the oligo isintegrated in the last replication fork, the efficiency ofrecombination would be lower, because only the last copyof the chromosome will carry the desired mutation. It hasalso been reported that the probability of an oligo beingintegrated into the chromosome is highly dependent onthe oligo, location on the chromosome, and how closely itis to another oligo that has been integrated [38, 39]. There-fore, when multiple oligos are used in a single electropo-ration event, it is difficult to predict the probability of eacholigo being integrated into the chromosome because theyare not mutually exclusive events.

4 Conclusion

The use of bacteriophage proteins to enable high-effi-ciency homologous recombination in E. coli has laid thegroundwork for the field of genome engineering, allowingfast and efficient editing of the genome at a much largerscale. Since the first report of recombineering in 1998 [1,3], several groups have focused their attention on improv-ing the system to achieve even higher efficiencies. In try-ing to optimize recombineering efficiencies, the mecha-nism of recombineering has been elucidated for theLambda Red system [28]. The design of the DNA cassetteitself was optimized: ssDNA oligos should be 60- to 90-mer, the 4 bases on the 5’ end should be phosphothionat-ed, and the oligo should be designed to target the laggingstrand. Multiple modifications to the replisome alsoincreased efficiency, knocking out the MMR by removingMutS, removing endogenous nucleases, and slowingdown DNA primase. Finally, coselection MAGE allowsresearchers to screen far fewer colonies for the desiredmutation based on the use of easily selectable/screenablecoselection markers, which is even more advantageousfor mutations that confer no obvious phenotype, such aspromoter- or ribosome-binding site changes.

One area that has yet to be optimized is concernedwith the issue of multiple chromosomes. Depending onthe medium, growth stage, and nutrient state of the cell,E. coli can have up to 16 chromosomes. Thinking aboutrecombineering efficiencies on a chromosomal basisinstead of a cellular basis can thus explain reduced over-all efficiencies. Colonies plated after short recovery peri-ods (30 min) will be sectored and require several more iso-lations or longer outgrowth to obtain a homogeneous pop-ulation of mutants. If the desired mutation confers adefect in growth, longer recovery times will dilute themutant population significantly. Growth in minimal medi-um can reduce the number of chromosomes per cell, butcells grown minimal medium are much less efficient attaking up DNA. Synthetic biology approaches can also beused to more finely control chromosome copy number; thefirst obvious target is DnaA. Experimental approachescan also be used to reach homogeneity faster. For exam-



ple, repeated recombineering cycles with the same oli-go(s) would reduce the number of generations to find puremutant colonies. Prior to efforts to reduce chromosomalcopy number, the ultimate goal of any experiment needsto be considered. Depending on the goal of the recombi-neering experiment, homogeneity may not be advanta-geous. If a homogeneous population is desired, cellseither need to be plated immediately and rescreened/selected until homogeneity is achieved or several roundsof recombineering can be performed with the same oligo(as in MAGE) to decrease the number of screens/selec-tions to find a homogeneous mutant. However, if the goalof recombineering is to obtain a library of cells with dif-ferent mixtures of mutations, then the presence of multi-ple genomes is in fact enabling the heterogeneity of thepopulation.

Recombineering has revolutionized the way we thinkabout engineering cells, enabling larger genome-wideediting. Many future applications of recombineering willbe focused on engineering entire pathways to optimizeexpression of all relevant proteins in a combinatorial way,so we can achieve higher product yields, as done forlycopene by Wang et al. [13]. Our current ability to engi-neer entire pathways and investigate combinatoriallibraries of expression levels is limited by the sheer size oflibraries required; increasing recombineering efficiencies(by reducing chromosomal copy number or any othermethod) would reduce the total number of cells that needto be screened to find a winning combination.

Funding for N.R. Boyle and T.S. Reynolds is provided byOPX Biotechnologies Inc.

The authors declare no conflict of interest.

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Professor Ryan Gill received his B.S in

chemical engineering from The Johns

Hopkins University in 1993; his M.S. in

chemical engineering from University

of Maryland College Park in 1997; his

Ph.D. in chemical engineering from the

University of Maryland College Park in

1999; and did postdoctoral work in

chemical engineering at MIT from 1999

to 2001. He joined the Chemical and Biological Engineering Depart-

ment at University of Colorado as an Assistant Professor in 2001,

where he is currently an Associate Professor. He is the Managing

Director of the Colorado Center for Biorefining and Biofuels and his

research focuses on the development of tools that enable high-effi-

ciency genome editing.



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Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing

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