Conditional DNA repair mutants enable highlyprecise genome engineeringAkos Nyerges1 Balint Csorgo

001 Istvan Nagy2 Dora Latinovics2 Bela Szamecz1

Gyorgy Posfai1 and Csaba Pal1

1Synthetic and Systems Biology Unit Institute of Biochemistry Biological Research Centre of the HungarianAcademy of Sciences Szeged H-6726 Hungary and 2Symbiosis and Functional Genomics Unit Institute ofBiochemistry Biological Research Centre of the Hungarian Academy of Sciences Szeged H-6726 Hungary

Received November 25 2013 Revised January 10 2014 Accepted January 11 2014

ABSTRACT

Oligonucleotide-mediated multiplex genomeengineering is an important tool for bacterialgenome editing The efficient application of thistechnique requires the inactivation of the endogen-ous methyl-directed mismatch repair system that inturn leads to a drastically elevated genomicmutation rate and the consequent accumulation ofundesired off-target mutations Here we present anovel strategy for mismatch repair evasion usingtemperature-sensitive DNA repair mutants andtemporal inactivation of the mismatch repairprotein complex in Escherichia coli Our methodrelies on the transient suppression of DNA repairduring mismatch carrying oligonucleotide integra-tion Using temperature-sensitive control ofmethyl-directed mismatch repair protein activityduring multiplex genome engineering we reducedthe number of off-target mutations by 85 concur-rently maintaining highly efficient and unbiasedallelic replacement

INTRODUCTION

Recent breakthroughs in genome engineering have greatlyexpanded our ability to design organisms in a directed andcombinatorial manner The myriad of novel genome-scalemodification technologies offer new opportunities for theconstruction of biological systems with desired properties(1) From these techniques oligonucleotide (oligo)-mediated allelic replacement has been optimized towardmultiplexing and automation (2) Specifically multiplex-automated genome engineering (MAGE) is capable ofediting and evolving the genome of a desired organism

Several factors make MAGE exceptionally attractivefor cell manipulation It enables simultaneous and cost-efficient modification of a large set of genomic targets andaccordingly exploration of the effects of mutations in acombinatorial manner by generating a diverse heterogenicpopulation across the targeted loci The power of MAGEhas been demonstrated in a wide range of biotechnologicalapplications It allows (i) optimization of metabolicpathways to produce industrially relevant compounds(23) (ii) improvement of bacterial growth propertiesunder selected conditions (4) and (iii) genome-wide re-placement of a specific codon in Escherichia coli (56)Therefore MAGE has the potential to transform basicresearch by accelerating and expanding the range ofprotocols for genome editing and analysis (1)MAGE relies on the incorporation of synthetic

single-stranded DNA (ssDNA) oligonucleotides carryingthe desired modifications into the lagging strand of thereplicating target genome (78) The efficiency of thisprocess is highly dependent on the avoidance of themethyl-directed mismatch repair (MMR) machinery ofthe target cell The removal of the endogenous MMRsystem increases the rate of oligo incorporation byorders of magnitude and eliminates biases in the rate ofincorporation of different types of mismatches (9)However the need for a disabled MMR machinery alsopresents the greatest drawback global MMR inactivationresults in an 100-fold increase in host mutation rate (10)leading to the accumulation of unwanted backgroundmutations across the bacterial genome For example inan effort to replace all TAG stop codons in E colithree different strains were sequenced after 25ndash30 cyclesof MAGE and subsequent genome transfer All of themwere found to carry gt100 off-target mutations (5)The severity of this problem is evident these off-targetmutations can potentially mask the phenotypic effects ofthe engineered modifications Several previous approaches

To whom correspondence should be addressed Tel +36 62 599 661 Fax +36 62 433 506 Email csabapalbrcmtahu

The authors wish it to be known that in their opinion the first two authors should be regarded as Joint First Authors

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The Author(s) 2014 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) whichpermits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited

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have been proposed to solve this problem (911ndash13)(Table 1) but all suffer from serious limitations aretechnically challenging or introduce novel methodologicalproblemsThe elimination of MMR activity is only necessary

during a relatively brief period of each MAGE cycle(ie during allelic replacement) Therefore we suggest asimple strategy relying on temperature-based control ofMMR protein activity In contrast to the relatively slowprocess of transcriptional control of MMR protein levelour protocol applies MMR protein variants withtemperature-sensitive defects allowing rapid switchingbetween mutator and nonmutator states thus minimizingthe time the bacterial population spends susceptible to theaccumulation of off-target mutations This temporal onndashoff switch of mismatch repair is easily incorporated intothe standard MAGE protocol as it already uses atemperature shift to activate the expression of the Redrecombinase enzymes

MATERIALS AND METHODS

Media chemicals and reagents

Unless otherwise noted cultures were grown in Luria-Bertani-Lennox (LBL) media (10 g of tryptone 5 g ofyeast extract 5 g of sodium chloride per 1L of water)for cell manipulations For the isolation of singlecolonies for further analysis cells were sampled on LBL

media supplemented with 15 agar and tetracycline at aworking concentration of 20 mgml For the measurementof rpsL allelic replacement frequency LBL agar wassupplemented with 50 mgml streptomycin LacZ MalKand AraB activities were assayed on MacConkey agarbase (peptone 20 g bile salts 15 g sodium chloride 5 gagar 135 g neutral red 003 g and crystal violet 10mgper 1 L of water) supplemented with 20 mgml tetracyclineand 1 of lactose for LacZ maltose for MalK orL-arabinose for AraB activity measurementsGlycerol-free terrific broth (TB) was applied for recovery

media (yeast extract 24 g tryptone 12 g K2HPO4 94 gKH2PO4 2 g per 1L of water)

Oligonucleotides

All oligonucleotides for allelic replacement as well aspolymerase chain reaction (PCR) primers used in thisstudy are presented in Supplementary File S1 Oligoswere ordered with standard purification and desaltingfrom Integrated DNA Technologies Oligos applied forallelic replacement have complementary sequence to thereplicating lagging strand and have minimized secondarystructure (gt12 kcalmol1) Additionally each oligocontained two subsequent phosphorothioate linkages atboth 50 and 30 termini for endogenous nuclease evasion

Strain construction

All applied strains were derived from E coli K-12MG1655 For the construction of temperature-sensitivemismatch repair deficient strain E coli tMMR previouslydescribed temperature-dependent mutS(A134V) andmutL(G62S) alleles (14) were introduced into wild-typebackground

Single-nucleotide variation in mutS which confers anAla134Val amino acid change was constructed byusing a suicide plasmid-based method Standard stepsand plasmids (pST76A pSTKST) of the procedure havebeen described (15) Briefly using PCR primers carryingthe desired point mutation (Supplementary File S1) themutant mutS gene was constructed then cloned into thethermosensitive pST76A plasmid The plasmid constructwas then transformed into the cell where it was able tointegrate into the chromosome by way of a singlecrossover between the mutant allele and the correspondingchromosomal region The desired cointegrates wereselected using the antibiotic resistance carried on theplasmid at a nonpermissive temperature for plasmidreplication Next the pSTKST helper plasmid wastransformed then induced within the cells resulting inthe expression of the ISce-I meganuclease enzyme which

Table 1 Various approaches to achieve high allele replacement (AR) efficiency while minimizing mutator phenotype

General strategy Specific attempts Possible limitations

Modifications in the ssDNA oligo

CC mismatch in vicinity of targetmodification (11)

Introduce novel scars place sequence limitations onwhich genome modifications can be madeproblems involving codon bias (11)

Change of wobble position of twoneighboring codons of targetmodification (11)

Use of chemically modified analogsof the four base pairs (12)

Added cost and lesser availability of themodified bases possible toxic effects onthe host organism (12)

Diversion of MMR proteins

Use of adenine analog 2-aminopurineto titrate MutL protein (9)

10-fold less efficient than with MMR deficientstrains extra incubation time mutageniceffect of 2-aminopurine (9)

Co-electroportation of dsDNA oligocontaining mismatches (9)

No effect on AR efficiencies even when present in100-fold excess (9)

Control of MMR protein transcriptionReversible inactivation of MMR protein

coding gene using oligonucleotide (13)

Requires extra MAGE cycles for the enrichment of theintroduced modification mutator phenotype persistsduring entire cycles when the gene is turned off (13)

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cleaves the chromosome at the 18-bp recognition sitepresent in the integrated plasmid The resulting chromo-somal break is repaired by way of RecA-mediatedintramolecular recombination between the homologoussegments in the vicinity of the broken ends Therecombinational repair results in either a reversion to thewild-type chromosome or in a scarless allele replacementStrains carrying the desired point mutation weredistinguished by sequencing the given chromosomalregion resulting in the MG1655 tMutS strainWhole-genome sequencing of the constructed strainrevealed a synonymous undetected point mutation in themutS gene (1323CT) which does not result in aminoacid change and has no effect on the desired phenotype

Oligonucleotide-mediated singleplex Red recombin-ation was used to generate the temperature-dependentmutL(G62S) variant Gly62Ser in MG1655 tMutS Togenerate the mutL point mutation a plasmid-basediterative Red recombination protocol was used As theintegrated mismatch (GT chromosomal-to-synthetic) hashigh correction efficiency without MMR inactivation theallelic replacement was done at elevated temperature topartially inactivate MutS proteins during oligo incorpor-ation (experiment based on unpublished results) The tem-perature-sensitive mutS mutant strain MG1655 tMutScarrying the pBADabg Red expression plasmid (16)was applied In brief to perform allelic replacementcells were grown in 10-ml LBL from overnight starterculture at 38C 250 rpm to OD550 05ndash07 Redproteins were induced by the addition of L-arabinose at02 concentration for 30min For recombination cellswere pelleted 3800 rpm for 7min and washed twice inice-cold purified water (dH2O) resuspended in dH2Oand electroporated with oligo MutL35_SNP at 25mMfinal concentration Electroporated cells were allowed torecover in 10ml of LB at 38C until the culture reachedmid-logarithmic growth After two iterations cells wereplated on LBL agar plates Clones with desired mutationwere identified by allele-specific PCR

Briefly selected colonies were assayed in 25-ml colonyPCR reactions using DreamTaq DNA Polymerase(Thermo Scientific catalog number EP0702) inDreamTaq Buffer (includes 2mM MgCl2) and 200 mMdeoxyribonucleotide triphosphates (Thermo Scientificcatalog number R0192) 02 mM MutL35ASP_fw and _rvprimers and 05 ml of saturated bacterial culture For allelediscrimination the following PCR protocol was used heatinactivation and cell lysis at 96C for 5min 30 cycles(95C for 30 s 635C for 30 s and 72C for 40 s) andfinal extension for 5min at 72C Colonies containingthe desired point mutation was confirmed by sequencingthe target site in mutL PCR amplicons generated by theMutLseq_fw and MutL35ASP_rv primers in colony PCRwere sequenced using the MutLseq_fw primer forsequencing

Permanent mismatch repair deactivation was achievedby endogenous mutS gene removal by suicideplasmid-based scarless deletion and yielded E coli K-12MG1655 mutS

The heat shock-inducible genomic Red system wasP1 transduced from E coli LT521 MG1655 gal490

nadATn10 pgl8 [ cI857 (cro-bioA)] (courtesy ofDonald L Court National Cancer Institute FrederickMD USA) This prophage-based construct wasintegrated at the bioAbioB locus of the constructedstrains and allowed the induction of gam beta and exogenes by a brief heat shock at 42C Integration of theprophage construct into E coli tMMR yieldedMG-tMMR MG-mutS was created by prophageintroduction into E coli K-12 MG1655 mutS

MAGE cycling process

Individual colonies from freshly streaked overnight agarplates were inoculated into glass flasks containing 10-mlLBL aliquots supplemented with 20 mgml tetracycline andincubated in a shaking incubator at 250 rpm at 32C Onreaching OD550 055ndash065 flasks were moved to aprewarmed 42C shaking water bath for 15min 300 rpmto induce expression of gam beta and exo recombinasegenes Induced cells were then immediately chilled on icewith vigorous shaking for at least 5min Cells were madeelectrocompetent by washing and pelleting twice in 10mlof ice-cold dH2O at 3800 rpm for 7min in an Eppendorf5702 R centrifuge with swing-out rotor at 4C Finallycells were suspended in 160 ml of dH2O All cellmanipulations were performed on iceFor electroporation 40 ml of competent cells were mixed

with 1 ml of 100 mM oligos suspended in TE buffer forsingleplex allelic replacement to reach 25-mM ssDNAconcentration For six-plex allelic replacements 6 ml ofoligo mixture was added containing 1 ml of each oligo at25mM final concentration Cells were subjected toelectroporation in a prechilled 1-mm gap VWRSignature Electroporation cuvette (VWR catalognumber 89047ndash206) using a BioRad MicroPulserelectroporator using the following parameters 1800V25 mF 200 Five milliliters of prewarmed (36C) TBmedium was immediately added to the electroporatedcells and was transferred to culture flasks Cells wereallowed to recover for 60min either at 32 or 36Cdepending on the experiment and the applied strainFive milliliters room temperature LBL medium supple-mented with 20 mgml tetracycline was then added andcells were allowed to grow at 32C until mid-logarithmicstate at 250 rpm At this point cells were either subjectedto additional MAGE cycles or assayed for phenotype andgenotype analysis Overall three separate incubators wereused during each cycle each preset to the giventemperature allowing for immediate shifts in thetemperature The above MAGE cycling protocol wasapplied generally except when noted in the experimentdescription

Optimization of recovery time

To determine the optimal time interval that cells spend atrestrictive temperature during each MAGE cycle a singleallelic replacement was performed using LacZ_m10_v2looligo This oligo introduces a nonsense mutation by twoconsecutive mismatches into the lacZ gene and has highcorrection efficiency in the presence of the native MMRsystem For the measurement of the correlation between

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the time interval that cells spend at restrictive temperatureand allelic replacement efficiency after electroporationLacZ_m10_v2lo oligo were introduced intoMG-tMMR in 25-mM final concentration After electro-poration cells were resuspended in 15-ml prewarmed TBbroth and incubated at 36C One milliliter of aliquots wasremoved and placed into 32C shaking incubator every15min for overnight incubation at 250 rpm in glasstubes Allelic replacement efficiencies were determined byplating cells at appropriate dilution from overnight cultureto MacConkey media supplemented with 1 of lactose

Analyzing recombination

For the analysis of allelic replacement frequencies duringMAGE cycles two types of selection marker genes wereselected for targeting either to introduce nonsense ormissense mutations The first set of genes includinglacZ malK araB and rpsL were selected for easy visiblediscrimination of recombinant colonies on supplementedMacConkey agar plates based on sugar fermentationandor antibiotic resistance (rpsL which confers resistanceto streptomycin) The second set of genes consisting ofcycA and hisB were targeted by knock-out oligonucleo-tides to introduce nonsense mutations Recombinantcolonies from recombineering with the second gene setwere screened by using allele-specific PCR for allelediscriminationTargeted inactivation of LacZ activity by the incorpor-

ation of a nonsense mutation was assayed by plating cellsonto MacConkey agar supplemented with 1 lactose as acarbon source and 20 mgml tetracycline Cells were platedat appropriate dilutions in three replicates typically toyield 400ndash800 colonies per plate Plates were incubatedat 30C overnight Allelic replacement efficiencies werecalculated by dividing the number of white (LacZ-)recombinant colonies by the number of total coloniesFor assaying MalK and AraB activities the sameprotocol was applied with the exception of the use ofMacConkey media supplemented with either 1 ofmaltose or L-arabinose respectivelyRpsL allelic replacement frequency was measured by

plating cells in parallel onto LBL agar and LBL agarsupplemented with 50 mgml streptomycin RpsL allelicreplacement efficiencies were calculated by dividing thenumber of StrR colonies by the number of total colonieson LBL agar platesAs it has been reported previously E coli cells can

contain up to eight partial chromosomal copies duringexponential growth in rich media (17) Fast growingcells that have not undergone enough cell divisions tosegregate mutant and wild-type alleles thereforegenerate sectored colonies To overcome this scenariocells were allowed to grow overnight to early stationaryphase before plating

Allele-specific colony PCR

Allele-specific colony PCR was used to genotype clones tomeasure allelic replacement frequencies for cycA and hisBand to select for clones with desired numbers of allelicreplacements For rapid allele discrimination two sets of

primers were synthesized either targeting cycA or hisBFor each set one primer carried the correspondingmismatch at the 30 terminus whereas the other hadcomplete homology to the target region Only a clonecontaining the mutant allele generated a PCR productTo avoid nonspecific product formation during PCRthe optimal annealing temperature and cycling protocolwas determined by gradient PCR with annealing tempera-tures varied between 58 and 70C

The query strains were assayed in 25 ml of PCR reactionsusing DreamTaq DNA Polymerase (Thermo Scientific) inDreamTaq Buffer (includes 2mM MgCl2) and 200 mMdeoxyribonucleotide triphosphates (Thermo Scientific)02 mM forward and reverse primer and 05 ml of saturatedbacterial culture (produced either by diluting one bacterialcolony in 100ml of dH2O or growing cells to stationaryphase in LBL) For cycA allele discrimination the followingPCR cycles were used heat inactivation and cell lysis at96C for 5min 28 cycles (95C for 20 s 61C for 30 s and72C for 40 s) and final extension for 5min at 72C

For hisB alleles the optimal PCR protocol was the samewith the exception of annealing temperature which wasset to 68C

The PCR products were separated on 1 agarose geland were imaged by a BioDoc-ItTM BioImaging System(UVP)

Genotype analysis

Genotype analysis at selected loci of cells that underwentrecombineering was performed by three main methodswhich included allele-specific colony PCR Sanger DNAsequencing and whole-genome resequencing

Sanger sequencing

Genotype analysis at selected loci of cells undergoingmultiplex recombineering was performed by Sangercapillary sequencing in addition to allele-specific PCRas previously described Genotype of regions correspond-ing to the annealing sites for the allelic replacementoligonucleotides and its proximity were determined inthis manner In all 600ndash700-bp PCR amplicons coveringthe target sites of all targeted mutations were sequencedfrom individual colonies Amplicons were generated bycolony PCR purified using Viogene GelPCR DNAIsolation System (Viogene BioTek Corp catalognumber GP1002) and sequenced on a 3500 SeriesGenetic Analyzer (Life Technologies LT) Mutationswere identified by aligning sequence reads to the referenceE coli K-12 MG1655 genome (GenBank accessionnumber NC000913 version NC_0009132 GI49175990)

For the analysis of variation in the target sites ofMAGE oligos 96 mutants of both MG-mutS andMG-tMMR strains were sequenced across the targetsite of the cycA-AAAC oligo within the cycA gene afterundergoing 20 MAGE iterations each Individual coloniesfrom populations that had undergone MAGE cyclingwere plated and isolated on LBL plates Colony PCRusing the cycA1 and cycAASP_r primers was used toamplify the oligo target site and its surroundings ThePCR amplicons were purified using ZR-96 DNA

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Clean-up KitTM (Zymo Research catalog numberD4018) Capillary sequencing was performed byMacrogen Inc Amsterdam Netherlands via EZ-SeqDNA sequencing service using the cycA1 primer forsequencing The obtained sequences were aligned to theE coli K-12 substr MG1655 chromosome usingGenomics Workbench 601 (CLC Bio) Variationsmapped within the target site of the cycA_AAAC oligowere judged as within-site mutations

Whole-genome sequencing

Genomic off-target mutation rates were determined byanalyzing five 6-fold mutants from each MAGE-cycledpopulation and comparing their genome with thesequenced ancestral strains LacZ malK and araB tripleknock-out mutants harboring an rpsL StrR mutation wereselected by plating cells to MacConkey media basesupplemented with 05 lactose 05 maltose 05L-arabinose and 50 mgml streptomycin at 30C Selectedcolonies were then screened for harboring cycA and hisBmutations using allele-specific PCR Cell lines were thenconfirmed for containing the desired targeted mutationsby Sanger sequencing using primers listed inSupplementary File S1 Selected cell lines were subjectedto whole-genome resequencing and SNV and INDELanalysis was performed

Briefly genomic DNA was extracted from selectedE coli isolates (GenEluteTM Bacterial Genomic DNAkit Sigma-Aldrich catalog number NA2110) and thesubsequent library preparation was performed using the5500 SOLiD Fragment Library Core Kit (LT) Threemicrograms of purified bacterial genomic DNA wasfragmented by Covaris S2 System to 100ndash250 bp Thefragmented DNA was end-repaired and ligated to P1(50-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-30) and P2 (50-CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGACGGCCAAGGCG-30) adapters that provide the primary sequences forboth amplification and sequencing of the sample libraryfragments The P2 adapter contains a 10-bp barcodesequence which provided the basis for multiplexsequencing (5500 SOLiD Fragment Library BarcodeAdaptors LT) The templates were size-selected usingthe Agencourt AMPure XP system (Beckman Coulter)nick-translated using Platinum PCR Amplification Mixand the template library was quantitated by quantitativePCR using SOLiD Library TaqMan Quantitation Kit(LT) The templates were clonally amplified by emulsionPCR with P1 primer covalently attached to the beadsurface Emulsions were broken with butanol and emul-sion PCR beads enriched for template-positive beadsby hybridization with P2-coated capture beadsTemplate-enriched beads were extended at the 30 end inthe presence of terminal transferase and 30 beadlinker Beads with clonally amplified DNA were thendeposited onto a SOLiD Flowchip the slide was loadedinto a SOLiD 5500xl System (LT) and the 50-basesequences were obtained according to the manufacturerrsquosprotocol

Bioinformatics analysis of genome sequences

The obtained sequences from each strain were firsttrimmed to filter out low-quality reads that were lt50 bpThe remaining high-quality sequences from each strainwere then aligned to the E coli K-12 substr MG1655chromosome (GenBank accession number NC000913version NC_0009132 GI49175990) in color space usingGenomics Workbench 601 (CLC Bio) An averagecoverage of gt130-fold was accomplished for each strainThe maximum gap and mismatch count within a singleread was set to 2 with a minimum of four reads to call apotential variation before further analysis Variationsrepresented only in the MAGE evolved lines and not inthe constructed ancestrals (with the exception of muta-tions within the oligonucleotide target sites) were judgedas off-target mutations arising during MAGE cycles

Mutation rate measurements

Mutation rates to rifampicin resistance were estimatedusing fluctuation analysis as previously described (18)Briefly 12 tubes of 1-ml LBL were inoculated with 104

cells from each strain Cells were grown at the examinedtemperature until early stationary phase Appropriatedilutions were spread onto nonselective LBL agar platesas well as LBL agar plates containing rifampicin (100mgml) and incubated at 30C Colony counts were performedafter 24 or 48 h respectively The mutation rate wascalculated using the Ma-Sandri-Sarkar maximumlikelihood method (19) The calculations were performedusing the FALCOR web tool (20)

Estimating effects of temperature increase on growthparameters of MGj-tMMR

Growth rate measurements were obtained by growingreplicates of the ancestral strains E coli K-12 MG1655wild-type MG-tMMR and MG-mutS Brieflycultures (06ndash1 ml) of the studied strains incubated at30C until early stationary phase were inoculated into96-well shallow plates (30 replicates per strains) Eachwell contained 100-ml LBL medium Growth curves wererecorded by measuring OD600 every 7min for 24 h at theexamined temperature using a Biotek-automated platereader Growth rate relative to wild-type E coli K-12MG1655 was calculated from the obtained growthcurves following a reported procedure (2122)

RESULTS

Characterization of the temperature-sensitive MMRmutant

Using standard genome engineering techniques(see lsquoMaterials and Methodsrsquo section) we introducedtemperature-sensitive MMR protein alleles[MutS(A134V) and MutL(G62S)] (14) into E coli K-12MG1655 expressing the Red recombinases resulting inthe strain MG-tMMRUsing rifampicin resistance assay and subsequent

fluctuation analysis (18) we estimated the mutation ratein this strain across a range of temperature settings

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In agreement with published results (14) we observed an100-fold increase in mutation rate as the temperatureshifted from 32 to 38C (Figure 1A)To test the effect of this temperature shift (and conse-

quent disruption of the MMR machinery) on thereplacement efficiency of ssDNA oligos we worked witha set of previously designed oligos (12) These oligosintroduce all possible single base pair mismatches (AACC GG TT GA GT CA CT) at specific genomiclocations and by generating premature stop codons theyeither inactivate lacZ or malK The frequency of thecorresponding allelic replacement events was thus easilydetectable by colorimetric assays (see lsquoMaterials andMethodsrsquo section) We found that in six of the eightcases the MG-tMMR strain allowed efficient andmostly unbiased oligo incorporation comparable withthat achieved by the isogenic mismatch repair knockoutstrain MG-mutS (Figure 1B) Although the incorpor-ation rates of CA and GG mismatches were significantly

lower in the MG-tMMR strain than that inMG-mutS the observed rates were still one order ofmagnitude higher compared with the values seen in theMG strain Additionally we tested the allelic replace-ment efficiency of another set of oligos (12) whichintroduce mismatches of increasing lengths into lacZAgain the replacement efficiencies for 1 and 2 consecutivemismatches in both MG-mutS and MG-tMMRstrains were over an order of magnitude higher thanwhat was observed in the MG strain (Figure 1C) Weconclude that MG-tMMR and MG-mutS providesimilarly high allelic replacement efficiencies both in thecases of single nucleotide changes and small insertions

Design and optimization of the modified protocol

Based on these results we designed a slightly modifiedMAGE cycle (Figure 2) Steps of the procedure are asfollows (i) a brief heat-shock (42C 15min) that serves

Figure 1 (A) Mutation rate measurement of employed strains at various temperatures A rifampicin resistance assay was used to calculate mutationrates as described in lsquoMaterials and Methodsrsquo section Error bars represent 95 confidence intervals of pooled samples of two independentmeasurements of 12 parallel samples each (B) Allelic replacement efficiencies of oligos generating various types of single base pair modificationsin the chromosome designated by chromosomal baseoligo base in MG MG-mutS and MG-tMMR The efficiency of allelic replacement wasestimated by the number of mutant cells per total colony-forming units The values are the means of two independent measurements each error barsrepresent standard errors An AG (GA) mismatch was created two separate times using two different oligos (C)Allelic replacement efficiencies ofoligos generating modifications of increasing size in the genome of MG MG-mutS and MG-tMMR The values are the means of twoindependent measurements each error bars represent standard errors For details see lsquoMaterials and Methodsrsquo section

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for transcriptional activation of the Red enzymes in theoriginal procedure (2) and can simultaneously inactivateMMR proteins in MG-tMMR (ii) electrocompetent cellpreparation and (iii) transformation of the ssDNA oligosinto the cells (0C 30min) The Red Beta-mediatedincorporation of the synthetic oligonucleotides into thetarget genome occurs during (iv) the cell recovery period(36C 60min) during which the elevated temperatureensures MMR protein inactivity This step is followedby (v) outgrowth phase at a lowered incubation tempera-ture (32C 120ndash180min) During the last phase MMRprotein functionality is quickly restored and cells canreplicate with low background genomic mutation rates

The main novelty of this procedure is that the mutatorstate coupled to growth is restricted to a brief period(phase 4) The settings for the two crucial variablestemperature and length in phase 4 (36C and 60min)were determined by an optimization process Importantlyelevated temperature induces the expression of the toxic pL operon (23) necessary for Red Beta-mediated incorpor-ation of synthetic oligonucleotides Although the optimalinduction temperature of this operon is at 42C growthretardation due to leaky expression was observed at lowertemperatures as well all the way down to 37C (Figure 3)At 36C both MMR protein activity and pL operontranscription remained sufficiently repressed ensuringboth undetectable cellular toxicity and high allelic replace-ment efficiency (Figure 1A) Regarding the optimal time of

recovery at 36C the answer appears to be more complexWe tested the allele replacement efficiency of a test oligo-nucleotide at different recovery time lengths and found anear linear correlation between the two variables (Figure 4)However a longer recovery period has at least two draw-backs it extends the length of each MAGE cycle and theamount of time the population spends in a mutator stateSixty minutes recovery time was found to be a good com-promise as allele-replacement efficiency was sufficientlyhigh (Figure 4) and it simultaneously minimized thechance of accumulating off-target mutations We anticipatethat depending on the exact goals of future experiments theprotocol can be optimized further eg by the shortening ofthe recovery period (phase 4) The results indicatethat the omission of phase 4 caused only a 40 decreasein the incorporation efficiency of an otherwise efficientlyMMR-corrected 2-bp mismatch (Figure 4)

Estimating the reliability of the improved MAGE protocol

To compare the performances of the original and theimproved protocols we carried out 20 cycles of MAGEusing MG-mutS and MG-tMMR populationsevolving in parallel In the case of MG-tMMR theMAGE protocol was modified as described above Weused synthetic oligos that introduce various types ofmismatches and target six different genes distributedwidely across the genome (Supplementary Figure S1)

Figure 2 General scheme of the modified MAGE protocol ssDNA oligos are incorporated into the bacterial genomes in a cyclical manner Theorange arrow represents the main novelty of the modified procedure a recovery period at 36C to which the mutator state is restricted to See maintext for details Adapted from Wang et al (2)

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Allelic-replacement efficiencies for each of the six oligoswere evaluated after 10 and 20 cycles of MAGE (Table 2)The high rate of allelic replacements observed inMG-mutS was mostly reproduced in MG-tMMRthe only exceptions being the araB AA and lacZ TTtransitions Even though allelic replacement efficienciesafter 20 cycles of these two oligos were significantlylower in the MG-tMMR strain the method is stilladequate in introducing these two types of mutationsinto the target strain at high efficiencies (see Figure 1B)Errors are known to occur during allelic replacement duein part to defects arising from oligo synthesis (5) To inves-tigate whether this had a different effect in the two proto-cols we sequenced one of the targeted genes (cycA) in 96isolated parallel evolved clones after 20 cycles from bothstrains From MG-tMMR 6 of 96 sequenced clones hadwithin-site mutations in regions corresponding to the an-nealing site of the 90-mer MAGE oligo cycA_AAACwhereas 9 of 96 clones from MG-mutS carried suchmutations The difference in the number of erroneousreplacements is not significant [X2 (2 N=192)=065P=042] The principal errors in the MG-mutSclones were single base deletions The majority ofmutations observed in MG-tMMR were nucleotidechanges but 4 of 5 originated from a specific change ofCT This is most likely linked to synthesis error duringoligonucleotide production (data not shown) Weconclude that the two constructs exhibit similarly lowerror rates and high efficiency of allelic replacementsNext we investigated the accumulation of off-target

mutations After 20 cycles of MAGE we selected fiveindependently evolved clones derived from bothMG-tMMR and MG-mutS All of them wereverified to carry all six specific targeted allelereplacements To infer the number of off-target mutationsthe genomes of these clones were sequenced using theSOLiD 5500xl System The sequenced clones derivedfrom MG-mutS carried 97 off-target mutations(single-nucleotide polymorphisms and indels) affecting

76 protein-coding genes (on average 194 point mutationsper clone) In contrast the clones derived fromMG-tMMR accumulated only 15 mutations (3 muta-tions per clone) Based on these figures we estimate thatthe improved MAGE protocol reduces the number ofoff-target mutations by 85 As expected (24) mostof the extra mutations in MG-mutS-derived cloneswere ATGC and GCAT transitions and theywere nearly completely absent in MG-tMMR-evolvedclones (Table 3) For a full list of all off-target mutationssee Supplementary Table S1

DISCUSSION

Techniques based on oligonucleotide-mediated allelicreplacement have several properties that make themespecially promising for genome-scale engineeringprojects (25) Allelic replacement is a general mechanismthat allows for the targeted modification of genes withoutthe need for restriction endonuclease sites antibioticselection of integrated constructs or the generation ofunwanted lsquoscarsrsquo in the vicinity of the target modificationAdditionally the high transformation efficiency and lowcost of single-stranded DNA oligos allow for thesimultaneous modification of multiple targets within asingle cell The MAGE method has been used to achievea number of key innovations including the generation of aso-called genomically recoded organism constructed bythe genome-wide replacement of a specific codon (6)However this recent breakthrough also pointed out themajor drawback of the technique Besides the targeted 321modifications 355 unwanted off-target mutations weredetected owing to the applied DNA repair-deficientcellular background (6) These undesired mutationscaused a reduction in fitness in the engineered strainsCorrection of particularly disadvantageous off-targetmutations is a painstaking approach that presents the

Figure 4 Optimization of recovery time at 36C Allelic replacementefficiency of a representative oligo that introduces a nonsense mutationby two consecutive mismatches in lacZ Values shown are the mean offour independent experiments error bars represent 95 confidenceintervals

Figure 3 Effects of temperature increase on growth parameters ofMG-tMMR Relative growth rate of MG-tMMR compared withwild-type E coli MG1655 (wild-type=1) at gradually elevatingtemperatures Values shown are the mean of 30 replicates each errorbars represent 95 confidence intervals

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added possibility of generating additional off-targetmutations Therefore it is apparent that future genomeengineering endeavors will need a more precise in vivogenome-editing approach with reduced off-target effectsto achieve increased genome stability

In this work we described a novel strategy with an aimto concurrently maintain low genomic mutation rate andhigh allelic replacement efficiency during multiplexgenome engineering We used temperature-sensitiveMMR protein variants to decrease background mutationsby temporal inactivation of DNA repair activity duringgenome editing Compared with the traditional workflowour protocol decreased the number of off-target mutationsby 85 Importantly this result was achieved withoutsignificantly decreasing the efficacy and accuracy ofoligo-mediated allelic replacement or the speed of theMAGE cycles Moreover the protocol is simple and can

be easily automated in the laboratory it only requires ashift in incubation temperature during cell recovery andthe use of a modified strain Furthermore our generalguideline of temporal inactivation of MMR may beapplicable to genome engineering of other organisms(26ndash31) Notably transient downregulation of MMRproteins using RNA interference to allow for oligonucleo-tide-mediated gene targeting has been demonstrated inmurine embryonic stem cells (3233) Importantlyoligonucleotide-mediated recombination engineering hasrecently been adapted for yeast (Saccharomyces cerevisiae)(34) and mismatch repair protein variants with tempera-ture-sensitive defects have been found in the same species(35) In summary our work improves the precision ofoligonucleotide-mediated genome engineering therebyallowing for more predictable and highly efficient cellprogramming for synthetic biological and industrialapplications

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

The authors thank Donald Court for providing the LT521strain and Balazs Horvath for aligning the SOLiD reads tothe reference genome

FUNDING

European Research Council (to CP) Wellcome Trust(to CP) Lendulet Program of the Hungarian Academyof Sciences (to CP) Hungarian Research Fund [OTKAPD 109572 to BC] Funding for open access chargeWellcome Trust

Conflict of interest statement None declared

REFERENCES

1 EsveltKM and WangHH (2013) Genome-scale engineering forsystems and synthetic biology Mol Syst Biol 9 641

Table 2 Allelic replacement efficiencies of all six used oligos in MG-mutS and MG-tMMR after 10 and 20 MAGE cycles

Allelic replacement ratio ()

Gene (mismatch type)(chromosomal base oligo base)

10 cycle population 20 cycle population

MG-mutS MG-tMMR MG-mutS MG-tMMR

malK (CC) 5208 3854 6875 7188cycA (AAAC) 5520 4791 8542 8854araB (AA) 4062 2395 5833 2917lacZ (TT) 7291 6666 9479 7188hisB (GT) 5937 6250 8958 8958rpsL (AC) 6041 6354 8542 7604

Fisherrsquos exact test was performed comparing each value of the MG-tMMR strain with the corresponding value of the MG-mutS strainPlt 005 Plt 001

Table 3 Number and types of off-target mutations observed in the

genomes of MG-mutS and MG-tMMR-derived strains after 20

MAGE cycles

Types of off-target mutations

MG-mutS MG-tMMR

Number Fraction Number Fraction

TransitionsATgtGC 34 351 3 20GCgtAT 52 536 9 60

TransversionsATgtTA 0 0 1 67ATgtCG 0 0 0 0GCgtTA 0 0 1 67GCgtCG 0 0 0 0

Number of insertions 8 82 1 67Number of deletions 3 31 0 0Total 97 15Consequences of substitutions

PositionNoncoding 14 144 2 133Coding 83 856 13 867

Within coding sequencesSynonymous 21 253 1 77Nonsynonymous 62 747 12 923

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2 WangHH IsaacsFJ CarrPA SunZZ XuG ForestCRand ChurchGM (2009) Programming cells by multiplex genomeengineering and accelerated evolution Nature 460 894ndash898

3 WangHH KimH CongL JeongJ BangD andChurchGM (2012) Genome-scale promoter engineering bycoselection MAGE Nat Methods 9 591ndash593

4 SandovalNR KimJYH GlebesTY ReederPJAucoinHR WarnerJR and GillRT (2012) Strategy fordirecting combinatorial genome engineering in Escherichia coliProc Natl Acad Sci USA 109 10540ndash10545

5 IsaacsFJ CarrPA WangHH LajoieMJ SterlingBKraalL TolonenAC GianoulisTA GoodmanDBReppasNB et al (2011) Precise manipulation of chromosomesin vivo enables genome-wide codon replacement Science 333348ndash353

6 LajoieMJ RovnerAJ GoodmanDB AerniH-RHaimovichAD KuznetsovG MercerJA WangHHCarrPA MosbergJA et al (2013) Genomically recodedorganisms expand biological functions Science 342 357ndash360

7 EllisHM YuD DiTizioT and CourtDL (2001) Highefficiency mutagenesis repair and engineering of chromosomalDNA using single-stranded oligonucleotides Proc Natl Acad SciUSA 98 6742ndash6746

8 YuD SawitzkeJA EllisH and CourtDL (2003)Recombineering with overlapping single-stranded DNAoligonucleotides testing a recombination intermediate Proc NatlAcad Sci USA 100 7207ndash7212

9 CostantinoN and CourtDL (2003) Enhanced levels of Red-mediated recombinants in mismatch repair mutants Proc NatlAcad Sci USA 100 15748ndash15753

10 SchaaperRM and DunnRL (1987) Spectra of spontaneousmutations in Escherichia coli strains defective in mismatchcorrection the nature of in vivo DNA replication errors ProcNatl Acad Sci USA 84 6220ndash6224

11 SawitzkeJA CostantinoN LiX ThomasonLCBubunenkoM CourtC and CourtDL (2011) Probing cellularprocesses with oligo-mediated recombination and using theknowledge gained to optimize recombineering J Mol Biol 40745ndash59

12 WangHH XuG VonnerAJ and ChurchG (2011) Modifiedbases enable high-efficiency oligonucleotide-mediated allelicreplacement via mismatch repair evasion Nucleic Acids Res 397336ndash7347

13 CarrPA WangHH SterlingB IsaacsFJ LajoieMJXuG ChurchGM and JacobsonJM (2012) Enhancedmultiplex genome engineering through co-operativeoligonucleotide co-selection Nucleic Acids Res 40 e132

14 HongES YeungA FunchainP SlupskaMM and MillerJH(2005) Mutants with temperature-sensitive defects in theEscherichia coli mismatch repair system sensitivity to mispairsgenerated in vivo J Bacteriol 187 840ndash846

15 FeherT KarcagiI Gyo00

rfyZ UmenhofferK Csorgo00

B andPosfaiG (2008) Scarless engineering of the Escherichia coligenome Microbial Gene Essentiality Protocols and BioinformaticsSpringer pp 251ndash259

16 MuyrersJPP ZhangY TestaG and StewartAF (1999)Rapid modification of bacterial artificial chromosomes byET-recombination Nucleic Acids Res 27 1555ndash1557

17 SergueevK CourtD ReavesL and AustinS (2002) E colicell-cycle regulation by bacteriophage lambda J Mol Biol 324297ndash307

18 FosterPL (2006) Methods for determining spontaneous mutationrates Methods Enzymol 409 195ndash213

19 SarkarS MaWT and SandriG (1992) On fluctuation analysisa new simple and efficient method for computing the expectednumber of mutants Genetica 85 173ndash179

20 HallBM MaC-X LiangP and SinghKK (2009) FluctuationAnaLysis CalculatOR a web tool for the determination ofmutation rate using Luria-Delbrck fluctuation analysisBioinformatics 25 1564ndash1565

21 WarringerJ and BlombergA (2003) Automated screening inenvironmental arrays allows analysis of quantitative phenotypicprofiles in Saccharomyces cerevisiae Yeast 20 53ndash67

22 WarringerJ EricsonE FernandezL NermanO andBlombergA (2003) High-resolution yeast phenomics resolvesdifferent physiological features in the saline response Proc NatlAcad Sci USA 100 15724ndash15729

23 YuD EllisHM LeeE-C JenkinsNA CopelandNG andCourtDL (2000) An efficient recombination system forchromosome engineering in Escherichia coli Proc Natl Acad SciUSA 97 5978ndash5983

24 LeeH PopodiE TangH and FosterPL (2012) Rate andmolecular spectrum of spontaneous mutations in the bacteriumEscherichia coli as determined by whole-genome sequencing ProcNatl Acad Sci USA 109 E2774ndashE2783

25 WangHH and ChurchGM (2011) Multiplexed genomeengineering and genotyping methods In VoigtC (ed)Methods in Enzymology Vol 498 Elsevier San Diego CApp 409ndash426

26 YamamotoT MoerschellRP WakemLP Komar-PanicucciSand ShermanF (1992) Strand-specificity in the transformation ofyeast with synthetic oligonucleotides Genetics 131 811ndash819

27 YamamotoT MoerschellRP WakemP FergusonD andShermanF (1992) Parameters affecting the frequencies oftransformation and co-transfromation with syntheticoligonucleotides in yeast Yeast 8 935ndash948

28 SwingleB MarkelE CostantinoN BubunenkoMGCartinhourS and CourtDL (2010) Oligonucleotiderecombination in gram-negative bacteria Mol Microbiol 75138ndash148

29 van PijkerenJ-P and BrittonRA (2012) High efficiencyrecombineering in lactic acid bacteria Nucleic Acids Res 40 e76

30 BrachmanEE and KmiecEB (2003) Targeted nucleotide repairof cyc1 mutations in Saccharomyces cerevisiae directed bymodified single-stranded DNA oligonucleotides Genetics 163527ndash538

31 DekkerM BrouwersC and te RieleH (2003) Targeted genemodification in mismatch-repair-deficient embryonic stem cellsby single-stranded DNA oligonucleotides Nucleic Acids Res 31e27

32 AartsM DekkerM de VriesS van der WalA and te RieleH(2006) Generation of a mouse mutant by oligonucleotide-mediatedgene modification in ES cells Nucleic Acids Res 34 e147

33 DekkerM de VriesS AartsM DekkerR BrouwersCWiebengaO de WindN CantelliE TonelliR and te RieleH(2011) Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotidesMutat Res 715 52ndash60

34 DiCarloJE ConleyAJ PenttilaM JanttiJ WangHH andChurchGM (2013) Yeast Oligo-Mediated Genome Engineering(YOGE) ACS Synth Biol 2 741ndash749

35 ArguesoJL SmithD YiJ WaaseM SarinS and AlaniE(2002) Analysis of conditional mutations in the Saccharomycescerevisiae MLH1 gene in mismatch repair and in meiotic crossingover Genetics 160 909ndash921

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