Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria

University of Groningen

Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid

bacteria

Bron, Peter A; Marcelli, Barbara; Mulder, Joyce; van der Els, Simon; Morawska, Luiza P;

Kuipers, Oscar P; Kok, Jan; Kleerebezem, Michiel

Published in:

Current Opinion in Biotechnology DOI:

10.1016/j.copbio.2018.09.004

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Bron, P. A., Marcelli, B., Mulder, J., van der Els, S., Morawska, L. P., Kuipers, O. P., Kok, J., &

Kleerebezem, M. (2019). Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria. Current Opinion in Biotechnology, 56, 61-68.

https://doi.org/10.1016/j.copbio.2018.09.004

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Renaissance

of

traditional

DNA

transfer

strategies

for

improvement

of

industrial

lactic

acid

bacteria

Peter

A

Bron

,

Barbara

Marcelli

,

Joyce

Mulder

,

Simon

van

der

Els

,

Luiza

P

Morawska

,

Oscar

P

Kuipers

,

Jan

Kok

and

Michiel

Kleerebezem

Theever-expandinggenomicinsightinnaturaldiversityof

lacticacidbacteria(LAB)hasrevivedtheindustrialinterestin

traditionalandnaturalgeneticmobilizationmethodologies.

Here,wereviewrecentadvancesinhorizontalgenetransfer

processesinLAB,includingnaturalcompetence,conjugation,

andphagetransduction.Inaddition,weenvisionthe

possibilitiesforindustrialstrainimprovementarisingfromthe

recentdiscoveriesofmolecularexchangesbetweenbacteria

throughnanotubesandextracellularvesicles,aswellasthe

constantlyexpandinggenomeeditingpossibilitiesusingthe

CRISPR-Castechnology.

Addresses

1_{BE-Basic,Delft,TheNetherlands} 2_{NIZO,Ede,TheNetherlands} 3_{RUG,Groningen,TheNetherlands} 4

HostMicrobeInteractomicsGroup,WageningenUniversity, Wageningen,TheNetherlands

Correspondingauthor:Bron,PeterA(peter.bron@nizo.com)

CurrentOpinioninBiotechnology2019,56C:61–68

ThisreviewcomesfromathemedissueonFoodbiotechnology EditedbyRuteNevesandHerwigBachmann

https://doi.org/10.1016/j.copbio.2018.09.004

0958-1669/ã2018ElsevierLtd.Allrightsreserved.

Introduction

Thelacticacidbacteria(LAB)areofgreatimportancein industrialfermentationandareprobablybestknownfor theirrole inthedairyindustry,but certainlyalsoplaya keyroleinavarietyoffermentationprocessesusingother food-raw materialsand feed-raw materials. Moreover, a continuouslyexpandingpanelofLABstrainsismarketed as health promotingprobiotics. An important industrial innovation strategy is theimprovement, expansion and diversificationof thestarterandprobioticculture reper-toire for the reliable production of healthy and tasty consumerproducts.Withthepresentcapacitiesin micro-bialgenomics,ourknowledgeofthemolecularbiologyof the LAB is rapidly expanding, providing us with an

unprecedented view of the diversity and evolution of these industrially important bacteria and exemplifying the evolutionaryimportance of horizontal genetransfer andmobilegeneticelements(MGEs)[1–3].Atan accel-eratingrate,wearediscoveringthecore-andpangenomes ofavarietyofindustriallyrelevantLABspecies,including isolatesoriginatingfromvariousenvironments(e.g.plant, intestine, etc.)or artisanal fermentation products. Such isolatesoftenencodephenotypesthatareofinterestfor industrial exploitation, such as stress robustness, flavor formation, bacteriocin production, substrate utilization, and bacteriophage resistance. Although comparative genomics, gene-trait matching and genetic engineering canestablishthegeneticbasisoftherelevantphenotypes, itisstillachallengetoharnessthesebiodiversity-derived discoveriesinindustrialstrainswithoutapplyinggenetic modificationmethodologies.Thishasinspiredarenewed interest in naturally occurring horizontal gene transfer processes,includingnaturalcompetence,phage transduc-tion and conjugation, for the mobilization of traits of interestto industrialstrains(Figure 1).

Natural

competence

Naturalcompetenceisacellularstateinwhichbacterial cells areable tointernalize exogenousDNA through a dedicated DNA uptake machinery that imports single strandedmaterial.Onceintracellular,thesinglestranded DNAisactivelystabilizedandsubsequentlymaintained asaplasmidorisincorporatedintothechromosome[4]. AmongtheindustrialLAB,naturalcompetencewasfirst established inthe yoghurt bacterium Streptococcus ther-mophilus in which formation of the quorum sensing complex ComRS results in expression of the master regulator of competence ComX [5]. The increased ComX level drivesthe expression ofthe DNA uptake machinery, a multiprotein complex composed of ComEA, ComEC, ComFA and ComFC, and several secondary competence proteins that facilitate DNA uptake (pilus-like structure proteins ComGA-GG) and protect internalized DNA (RecA, SsbA, SsbB, DprA). ThisstateofnaturalcompetencewasobservedwhenS. thermophiluswasgrowninchemicallydefinedmediumor whensyntheticpeptidesrepresentingtheC-terminalof ComSwereadded[6,7].Ithasbeenusedtotransferthe geneencodingtheextracellularproteasePrtPto proteo-lytically negative strains [8], and togenerate histidine prototrophyinstrainsauxotrophicforthisaminoacid[9].

Althoughthepresenceof(remnantsof)thecompetence genes was observed more than a decade ago in Lacto-coccuslactis[10],itwasonlyrecentlyshownthat moder-ateoverexpressionofcomXindeedresultedinthe asso-ciatedcapabilitytointernalizeDNA[11,12].Similarly, overexpressionofanalternativesigma factorledto the induction of competence genes in Lactobacillus sakei, although in this organism no transformation could be observedunderthe conditionstested [13].Toevaluate the phylogenetic conservation of this genotype among the lactobacilli, we evaluated the completeness of the genesetencodingtheDNAuptakemachineryinsubset ofLactobacillusgenomes(Table1),and concluded that forall ofthese species, strains could beidentified that encodeacompletegeneset,althoughinspecific (NCBI-reference)strainsoneormoreofthese genesappearto be disrupted by mutations.Although requiring experi-mentalvalidation, thisimplies thatthe natural compe-tence phenotype potentially can be activated in many different LAB, although the regulatory mechanisms underlying competence activation in these bacteria remains to beelucidated. Nevertheless, the broad dis-tribution of the genes required for the DNA uptake machinery may enable novel approaches towards gene-exchange and phenotype-exchange between strains. The acceptance of such strains in the food industry from a regulatory point of view would be tremendouslyaided bytheidentificationofthenatural conditions that trigger the uptake of DNA which are currently only established for specific S. thermophilus strains[9].

Conjugation

Conjugativeplasmidsaswellasintegrativeand conjuga-tive elements (ICEs) are vertically propagated during replication and cell division.These conjugative MGEs encodesimilartypeIVsecretionmobilization machiner-iesthatareinvolvedinoriT-dependentconjugaltransfer to appropriate recipient cells, but also encode distinct functionsinvolvedinchromosomalintegrationand exci-sion (ICEs), and extra-chromosomal replication (plas-mids) [14,15]. The genetically conserved functions of these conjugative MGEs have been exploited in tools aiming to detect them in bacterial genome sequences [16,17,18],whiledelimitation ofICEscanbeachieved by pan-genome and core-genome mapping [16] or by curing them from the host chromosome [19]. Besides theircanonicalfunctions,theconjugativeMGEsencodea variablenumberofaccessory genes(‘cargo’)thatconfer phenotypestohostcells[15,20].Sincetheircargo encom-passesanumberofrelevantindustrialtraits,conjugative MGEshavereceivedconsiderableattentioninLAB.For example,in L.lactis genes encoding lactose utilization, extracellular proteinase, and polysaccharide production are commonly encoded on conjugal plasmids [20], whereasnisinproductionaswellassucroseandraffinose utilizationareencodedonICEs[21,22].Mobilizationof theseelementsallowsthecombinationofbeneficialtraits inasinglestrain[23],oralterationofastrain’scapacityto interactwithitsenvironment[24].However,MGEshave alsobeenassociatedwithundesirabletraitslikeantibiotic resistance. This is particularly common among various streptococci, including S. thermophilus [25]. On the one

62 Foodbiotechnology

Figure1

PHAGE TRANSDUCTION CONJUGATION NATURAL COMPETENCE

Current Opinion in Biotechnology

Schematicrepresentationofthe3‘traditional’strategiesforgeneticmobilization.

DNA transfer strategies in lactic acid bacteria Bron et al. 63 Table 1

Natural competence in lactobacilli. The established genes encoding the DNA uptake machinery of Lactococcus lactis KF147 [12] were used to identify homologous genes in Lactobacillus plantarum WCFS1, identifying a complete late competence geneset in this species. Subsequently, the L. plantarum competence genes were used to search the genomes of a set of other Lactobacillus genomes (initially targeting the NCBI-reference genome for each species). When the Lactobacillus reference genome sequences contained disrupted competence genes (pseudogenes), it was evaluated whether other genomes of the same species contained intact versions of these genes. Notably, the comEB and comC genes are known to be absent or not expressed in lactococci that have been experimentally established to be able to become competent, which implies that these genes are not essential for competence development. Taken together the results of this in silico analysis indicate that in all Lactobacillus species evaluated here there are at least some representative strains that encode a complete geneset for the physical DNA uptake machinery

Protein length (nr. of residues)/protein sequence similarity (%a_{) compared to L. plantarum WCFS1 (or L. lactis KF147 in the case of L. plantarum WCFS1)}

Species strain ComC ComEA ComEB ComEC ComFA ComFC ComGA ComGB ComGC ComGD ComGE ComGF ComGG ComX

Lactococcus lactis KF147 221 NA 215 NA Absent NA 736 NA 440 NA 216 NA 312 NA 357 NA 127 NA 143 NA 98 NA 141 NA 94 NA 163 NA Lactobacillus plantarum WCFS1 226 39 241 54 161 NA 763 45 450 51 224 53 324 56 349 47 118 45 157 36 70 38 162 31 54 24 187 42 Comparative analysis with other Lactobacillus genomes, using the L. plantarum WCFS1 protein sequences

Lactobacillus rhamnosus GG Absent 221 52 Absent 734 47 421 53 223 53 289 55 317 44 106 38 140 39 103 32 155 31 107 24 179 44 Lactobacillus acidophilus NCFM 229 42 227 54 Absent 762 48 427 57 231 48 324 61 334 46 119 54 142 34 89 28 187 37 57 36 178 48 Lactobacillus paracasei ATCC 334 Absent 223 51 Absent Pseudogeneb _{420 54 222 51 288 56 317 43 107 39 146 38 106 33 153 33 110 29}

182 46

Lactobacillus salivarius UCC118 218 47 229 51 158 86 753 51 445 66 228 54 327 64 357 56 100 53 140 37 84 46 Pseudogeneb _{93 22 192 42}

Lactobacillus fermentum IFO 3956 241 46 224 53 159 83 745 57 438 67 224 56 319 62 327 49 105 58 161 40 98 34 147 43 79 37 192 45 Lactobacillus gasseri ATCC 33323 225 46 227 52 Absent 761 48 422 55 223 50 325 62 326 43 98 55 138 38 72 42 172 37 52 35 185 42 Lactobacillus helveticus CNRZ 32 227 45 231 51 Absent 762 48 428 57 231 48 324 61 333 43 116 51 143 31 89 33 166 38 58 38 181 47 Lactobacillus reuteri DSM DSM 20016 224 48 210 53 161 84 703 51 443 67 226 62 325 67 356 49 103 55 144 45 96 34 143 40 68 38 191 45 Lactobacillus sanfranciscensis TMW 1.1304 Pseudogeneb _{227 53 161 83 742 52 Pseudogene}b _{224 55 324 62 336 50 99 50 148 43 56 42 144 38 55 42 194 41}

NA = Not applicable.

a_{Needleman-Wunsch Global Align Protein Sequences tool in protein BLAST.} b_{The protein is encoded in the genome of other strains of the same species.}

cedirect.com Current Opinion in Biotechnology 2019, 56 :61–68

hand, transferof conjugativeMGEs appears to be con-strainedtoanMGE-specificrangeofcompatibleacceptor strains[26],whereasontheother handICEshavebeen reportedtobetransferableacrossthespeciesborder[27]. Intriguingly,ithasbeenproposedthatconjugative plas-midandICElifestylesofMGEsareinter-changeableand playdistinct roles in bacterial evolution,in which plas-midsdisplayincreasedgeneticplasticitybuthaveamore constrainedhost-rangethantheirICEcounterparts[28]. Taken together the conjugative MGEs often encode industriallyrelevanttraits,andgenomicscombinedwith dedicated search engines enables thediscovery of new conjugativeplasmidsand ICEs.Tobetter harnesstheir potentialinindustrialstrainimprovementapproachesitis importanttobetterunderstandtheirmechanismof trans-ferandthecognatehost-rangelimitations.Inthiscontext itisalsoimportanttobetterunderstandtheroleofgroup IIintrons,liketheonepresentintheL.lactissex-factor [29], in the modulation of transfer efficiencies of con-jugativeMGEs[30].

Bacteriophage

transduction

Bacteriophages are viruses that infect bacterial cells, hijacking thehost replication, transcription and transla-tion machineries to drive their proliferation. Bacterio-phagesinfectingLABhavebeenextensivelyinvestigated astheyrepresentoneofthemajorcausesoffermentation failureindairyfactories.Themajorityofphagesinfecting LAB belong to the Siphoviridae family, complemented withmembersoftheMyoviridaeand Podoviridaefamily, eachwithdistinctphagetailcharacteristics[31].Formost species within the Siphoviridae family, including the speciesmost frequently encounteredin thedairy envi-ronment(P335,936andC2[32]),panviromeshavebeen established[31,33].Twomainmodes ofpackaginghave been recognized, based on either cohesive ends (cos phages) or headful packaging (pac phages). The latter mode of packaging is initiated on a single recognition sequenceandterminatedwhenthephageheadisfull,a process that is proneto promiscuouspackaging of host DNA [31]. Plasmid or chromosomal genes involved in sugar fermentation, proteolysis or antibiotic resistance were transferred betweenLAB strains via phage trans-duction.High-frequency plasmidtransductionobserved inL.lactiswasexplainedbytheshorteningoftheoriginal plasmidtoasizethatexactlyfittedthephagehead[34]. Infection of a new host by bacteriophages has led to successfultransfer of bacterial DNAbetweenstrains of poorlygeneticallyaccessible organismssuch as Lactoba-cillus delbrueckii [35] or even between different LAB species [36]. However, host-specificity is dictated by thecombinationofphage-encodedreceptorbinding pro-teins(RBPs)thatassociatewiththephagebaseplateand thecellwallpolysaccharideand/orproteinaceous recep-tors on the host surface [37]. Even within the phage species 936 five RBPs have been identified [38],

showcasingthestrongconstraintsofphage-host recogni-tionthatcouldlimittheirpotential for genomic mobili-zation.However,thisnotioniscontrastedbythe demon-stration that plasmid transduction by a certain phage could be exploited for cross-species plasmid transfer betweenL.lactis andS.thermophilus[36].Another tech-nicalchallengelieswithinthefactthatonewouldneedto establish appropriate phage transduction protocols for each individual phage to prevent loss of the receptor populationduetophagepredation.

Despitetheseadvancesinourunderstandingofphagehost recognitiononlylimitedattentionhasbeengivento gen-eralizedgenome mobilizationbypromiscuouspackagingof thegeneticmaterialofthehostusedforphage-propagation. IdentifyingeffectivetransducingbacteriophagesinLAB couldopennovelapproachestowardsgenomicexchange between strains, which could be exploited to harness naturaldiversityfortheimprovementofindustrialstarter cultures,particularly if bacteriophagescan beidentified thatdisplayabroadhostspecificity.

Perspectives

Besides the revival of traditional methods described above, a few emerging technologies might also have potentialto enablenatural DNAtransferor couldallow dedicated genome editing and engineering in existing industrialstrains.

Nanotubesaretubularmembranousbridgesbetweencells forwhichevidence ismountingthattheymediate cyto-plasmicmolecular trade amongneighboring cellsof the same and different species [39]. For instance, plasmid transfer has been demonstrated from Bacillus subtilis to otherspeciesincludingStaphylococcusaureusandEscherichia coli[40].Moreover,B.subtilis hasbeenshownto inhibit BacillusmegateriumgrowththroughthedeliveryofatRNase toxinviananotubes,allowingnutrientextractionfromthe paralyzedcells[41].Thefactthatextracellularmembrane vesiclessharetheirmembranousnaturewiththenanotubes maysuggestthatthesecommunicationvehiclesalsoshare similar but currently not fully identified machineries involved in their production, with membrane vehicles fusingto anddissociatingfromnanotubes[39].Notably, extracellularvesiclesofvariousbacteriahavebeenshown tocontainDNA,metabolitesand/orproteinsandcanfuse withbothprokaryoticandeukaryoticcells[42,43].Thereby bothnanotubesandextracellularvesiclesprovidean enor-mouspotentialforthenaturaldistributionandexchangeof geneticmaterialandcognatephenotypesbetweenbacteria ofthesamespeciesaswellasacrossthespeciesborder[39]. Tothebestofourknowledge,therehavenotbeenreports ofnanotubesinLABtodate,andonly fewreportshave identified extracellular vesicles in different pathogenic streptococci [44–46] and some probiotic lactobacilli [47,48].Therefore,thismechanismofmolecularexchange

64 Foodbiotechnology

deservesmoreattentionintheLAB,toevaluateits poten-tialingenomemobilizationandgeneticexchange.

TheroleoftheCRISPR-Cassystemasabacterial adap-tive immune system involved in acquiring resistance against bacteriophageswas pioneered in S.thermophilus [49] and E. coli [50]. Ever since, CRISPR-Cas systems have beendiscovered in avarietyof bacteria,including severalLAB[51].Thecompositeanddynamicnatureof the CRISPR array has proven to be an efficient and practical target for the typing and tracking of bacterial strains, including industrial starter culturesand health-promotingprobioticstrains[52,53].Moreover,theroleof thesystemintheacquisitionof phageresistance canbe effectivelyemployedtoexpandphageresistanceprofiles in specificstrains[54].

TheCRISPR-Cassystemwasexploitedforthe construc-tionofaprogrammablegenomeeditingtoolbox,typically employing the Streptococcuspyogenes type-II Cas9 endo-nuclease(SpyCas9)[55].Cas9canbetargetedtoaspecific geneticsequencebyacomplementaryshortguidingRNA (sgRNA) provided that the sequence is flanked by the protospacer adjacent motif (PAM; NGG for SpyCas9). Recently, phage-assisted evolution allowed the adjust-mentofthePAM-specificityinSpyCas9derivatives[56], enabling the expansion of the sequences that can be targeted.Onceguidedtoitstargetlocus,Cas9introduces a double strand DNA break in the targeted DNA sequence,whichisthefoundationofitsimmunity func-tion that protects bacteria against exogenous DNA [49,50,55].The CRISPR-Cas9 toolbox has been exten-sivelyusedineukaryoteswherethedouble-strandbreaks introducedbyCas9canberepaired bynon-homologous endjoining(NHEJ),whichcreatesoutofframedeletions and insertions (INDELs), leading to gene disruption. Alternatively,thesedoublestrandbreakscanberepaired by homologous recombination (HR) when a ‘repair template’ is provided in parallel, allowing highly site-specific mutagenesis [57]. Bacteria commonly lack the NHEJcapacity,anddoublestrandDNAbreaksarelethal in most bacteria,which caused the application of Cas9 tools in bacteriato lag behind[19,55,58].Actually, the lethality of doublestrandDNAbreakswasexploitedin the curing of mobile genetic elements like prophages, plasmids, ICEsandgenomicislandsfromvarious bacte-ria, including LAB [19,59,60]. Bacteria do have an endogenous HR machinery, and the application of Cas9-sgRNA in combination with repair templates has proven to be effective in various bacteria, including several LAB and their phages [61–64]. Moreover, a Cas9derivative thatis catalyticallyinactivatedby point mutations (so-called deadCas9; SpyCas9D10A,H840A) has beenusedingenesilencingindifferentbacteria, includ-ing L.lactis[65].Recently,Cas9-base-editor fusion pro-teinswerereportedthatinsteadofintroducingadouble strand DNA break introduce a specific nucleotide

substitution in the targetsequence[66,67].Thisnext generationofCas9toolswillprobablyacceleratetheuse of thesemethodsinprokaryotesbecausetheyavoidthe requirement for a repairtemplate and enable effective genome-editing.

The extreme precision of the Cas9 editing approaches enables the highly effective construction of derivatives thatareidenticaltomutantsthatemergedspontaneously or were generated by random mutagenesis. Mutants constructed by CRISPR-Cas genetic engineering are indistinguishable from mutants produced by methods acceptablefor regulatorybodies, which could,or rather should,changelegislationperspectivesonthe classifica-tion of these derivatives as genetically modified organ-isms toensureenforceableand non-discriminatorylegal guidelines.Thisopinionhasalsobeenexpressedbythe lactic acid bacteria industrial platform (LABIP) after a dedicated workshopinMay 2017[68].

Concluding

remarks

Althoughseveralofthegenemobilizationstrategies dis-cussed here are considered ‘classical’ in experimental molecular microbiology, they are receiving renewed attentionbecauseoftheirpotentialtoenablethe capital-izationoftheexpansionofourknowledgeofthegenetic and phenotypic diversity among LAB. The application possibilitiesofthedifferentmobilizationstrategiesrange from generic genomic mobilization by natural compe-tence and generalized bacteriophage transduction, to dedicated mobilization of specific traits associatedwith conjugative MGEs. The latter category is known to encode a variety of industrially relevant traits and has traditionallybeen exploitedto improve startercultures, for example the construction of proteolytically active, nisin-resistant and nisin-producing, or polysaccharide producingstartercultures[69].Contrarytonatural com-petence, which is unrestricted by strain compatibility becauseitinvolvesimportofnakedDNAandprincipally allows thetransferof very largeDNA fragments,MGE conjugationandphagetransductionarerestrictedby host-rangelimitationsandenabletransferoffragmentsuptoa certainsize(definedbythephagepackagingcapacity,or theICEdelimitation).Ourknowhowofthesehostrange limitationsisrestrictedtorelativelyfewwell-established examples [20,37], and increasing our mechanistic understanding of strain-compatibility in conjugation shouldhelptoovercomesuchspecificity-borders. More-over,itcanbeanticipatedthatwithintheextreme diver-sityofphagerepertoiresinnature,theremaybe environ-mental phages with a much broader host-range as compared to those that have been studied to date on basisoftheirdetrimentalactivityinindustrial fermenta-tion.Expandingresearchtotypicalenvironmentalphages fromwastestreamsmayallowtheisolationofLABphages that do not cause any industrial problems,but may be

muchmore proneto accommodate experimental trans-ductionamongawidervarietyof strains.

Novelapproachesusingtheemergingpotentialof mem-branousconnectionsbetweenbacterialcells(withinand across species border), like nanotubes or extracellular vesiclesoffer excitingpossibilitiesfor genetic mobiliza-tion,althoughitremainstobeestablishedtowhatextent these processes are non-selective and can actually be employedforgenericmobilization.

Irrespectiveof thetransfertechnologyemployed, selec-tion of the acceptor strain that has incorporated and expressesthedesirednovelgenetictraitremainsa chal-lenge.Manyofthemostinterestingindustrialtraitsdonot allowphenotypicselection(e.g.flavorformationcapacity, specificexopolysaccharideproduction,etc.),andisolating improved strains-enriched with a non-selectable geno-typeremainschallengingandrequires extreme-through-putscreeningpossibilitiesthatmaybefacilitatedbythe developmentsinmicrofluidicsandemulsiontechnology [70,71,72]. Alternatively, it may be worth investing in strategiesthataimto enrichfor thegeneticlocithatare meant to be transferred prior to their actual transfer, simply to reducethedemand on thethroughputof the downstreamscreeningmodel.Employing(RING-)FISH single-moleculedetectionstrategies[73,74]requires fluo-rescent labelling of cells which is not compatible with post-selectionbacterialgrowth.Overcomingthis techno-logical hurdle deserves further attention, since such methodologies could facilitate high-throughput single-cell-based genetic screening and selection using flow-cytometryandsorting.

Finally,followingthecontinuouslyexpandingapplication of the CRISPR-Cas technology in eukaryotes, the recently emerging advances in this toolbox have over-come the initial problems of lethality of double-strand chromosomal nicks in bacteria and open tremendous possibilities for fine-grained strain improvement strate-gies offered by nucleotide-specific genome editing. At presentthestrainsresultingfromCRISPR-Casgenome editingwouldberegardedgeneticallymodified,butthe strong arguments of enforceability and non-discrimina-tionfavorreadjustmentoflegislationinthisarea, liberat-ingthesestrategiesfromthisconstraint,possiblywithan appropriate case by case evaluation regimen to allow surveillanceoftheengineeredorganisms.

Conflict

of

interest

statement

Nothingdeclared.

Acknowledgement

ThisworkwascarriedoutwithintheBE-BasicR&DProgram,whichwas grantedanFESsubsidyfromtheDutchMinistryofEconomicAffairs.

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