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Site-Specific Selfish Genes As Tools for the Control and Genetic Engineering

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Site-Specific Selfish Genes As Tools for the Control and Genetic Engineering Received 14October 2002 Accepted 12December 2002 Publishedonline 19March 2003 Site-specificselfish genes astools for the control andgenetic engineering of naturalpopulations Austin Burt Departmentof Biological Sciences and Centrefor Population Biology, Imperial College, Silwood Park,Ascot, BerkshireSL5 7PY,UK ([email protected] ) Site-specificselfish genes exploit hostfunctions to copy themselvesinto adefinedtarget DNA sequence, andinclude homing endonucleasegenes, group IIintronsand some LINE-like transposable elements.If suchgenes can be engineered to target newhost sequences, then they canbe usedto manipulate natural populations,even if thenumber of individuals releasedis asmall fraction ofthe entire population. For example, ageneticload sufficientto eradicate apopulation canbe imposed in fewerthan 20 generations, if thetarget is an essentialhost gene, the knockout is recessiveand the selfish gene has an appropriate promoter. There will beselection for resistance,but several strategies are available for reducingthe likeli- hoodof it evolving. Thesegenes may also beused to genetically engineernatural populations,by means ofpopulation-wide gene knockouts, gene replacements and genetic transformations. By targeting sex- linked loci justprior tomeiosis one may skewthe population sexratio, andby changing thepromoter onemay limit thespread of the gene to neighbouring populations.The proposedconstructs are evol- utionarily stable in theface of the mutations most likely toarise during their spread,and strategies are also available for reversing themanipulations. Keywords: population eradication; population geneticengineering; homing endonucleasegenes; vector-bornediseases 1. INTRODUCTION notbe useful. Whichever classof site-specific selfish gene is used,all thevarious proposals presupposethe ability to Somespecies— arelatively small number—cause substan- engineersuch genes to recognize a newtarget sequence. tial harm tothe human condition;most prominent are Work ondesigning enzymesto recognize aspecifiedDNA thosethat causedisease, transmit diseaseor reduceagri- sequenceis ongoing, motivated in part by their potential cultural output.Many such species have long beentargets usein functionalgenomics and gene therapy ofpopulation control,with varying degreesof success, but (Chandrasegaran &Smith 1999; Segal et al. 1999; Bibi- somespecies are still beyondcontrol by currentmethods, kova et al. 2001; Buchholz &Stewart2001; Chevalier et andnew approaches are required.Genetic methods of al. 2002; Santoro& Schultz2002; Seligman et al. 2002; engineering or eradicating natural populations have been Takahashi &Fujiwara 2002). Engineering group IIintrons muchdiscussed (Knipling 1979; Curtis1985; Hastings totarget newsequences is particularly simple, asrecog- 1994), mostrecently in thecontext of using transposable nition largely dependsupon RNA– DNA basepairing (Guo elementsor bacterial symbiontsto drive novel genesof et al. 2000). Despitethis activity, theuses of engineered interestinto apopulation (Ribeiro &Kidwell 1994; selfishgenes for manipulating natural populations appear Beerntsen et al. 2000; Braig &Yan2002). However,there notto be recognized, and an exploration ofthe possi- are inherentdifficulties with theseproposals, in particular bilities seemswarranted onthree grounds: to motivate relating tothe stability ofthe proposed constructs, and more rapid developmentof the technology; to warn of goodreasons to think they may notwork (Turelli & containmentissues that ought tobe addressed during Hoffmann1999; Braig &Yan2002; Spielman et al. 2002; development;and to stimulate discussionson the desir- and§ 6, below).In this paper Iexplore aseriesof alterna- ability oferadicating or genetically modifying particular tive geneticapproaches basedon the use of site-specific species. selfishgenes— genes that exploit hostfunctions to copy themselvesinto aparticular target sequence.These alter- 2. THE BASICCONSTRUCT native approaches appear tohave anumberof desirable features,including evolutionary stability andreversibility. HEGsare selfishor parasitic genesthat canspread Naturally occurring examples ofsite-specific selfish through populations owing totheir biased‘ super-Mendel- genesinclude homing endonucleasegenes (HEGs), group ian’inheritance (Chevalier &Stoddard2001; Goddard et IIintronsand some site-specific LINE-like transposable al. 2001). They encodean enzyme that recognizesand elements(Chevalier &Stoddard2001; Belfort et al. 2002; cleavesa 20–30 bp sequencefound on chromosomes not Eickbush2002). Outof the three types, HEGs have the containing acopy ofthe HEG. The HEGitself is inserted simplest mechanism ofaction (describedfurther in §2), in themiddle ofits ownrecognition sequence,and so while theother twospread via an RNA intermediate and chromosomescarrying theHEG are protectedfrom being reversetranscription. For simplicity ofexposition, HEGs cut.The brokenHEG 2 chromosomewill typically be will beused as exemplars throughout thepaper, though repaired by thecell’ s recombinational repair system,which this is notmeant toimply that theother twotypes will usesthe intact HEG 1 homologue asa template. After Proc.R. Soc.Lond. B (2003) 270, 921–928 921 Ó 2003 TheRoyal Society DOI10.1098/ rspb.2002.2319 922 A. Burt Site-specic selsh genes andpopulation control recognition site 1.0 s s 0.8 e n t i f essential gene r 0.6 o y c n 0.4 e u q e r f 0.2 essential HEG gene 0 10 20 30 40 50 time (generations) Figure 2. Frequency of theHEG (solid curve) and population mean fitness (dashed curve) assuming e = 0.9 and an initial release frequency of 1%. Theseresults, and all meiosis- or germline- no self-splicing others in thepaper, are for an idealized population, from specific promoter intron or intein whichall real populations will deviate in some way.They should, therefore, betaken as rough indications, not precise Figure 1. Aconstruct for biological control: aHEG predictions. engineered to recognize asequence in an essential gene for whichthe knockout phenotype is recessive. Note thatthe weassume that thepopulation is large andmates ran- HEGis inserted into themiddle of its own recognition sequence. domly, that theknockout is arecessivelethal andthat TRD occursequally in males andfemales, then the equi- librium frequencyof the HEG ( qˆ)canbe shown to be repair, both chromosomeswill containa copy ofthe HEG, qˆ = e, where e is theprobability that theHEG 2 allele in a anda heterozygote will have beenconverted into ahomo- heterozygote is convertedto aHEG 1 allele; e = 0 for Men- zygote. Thus,the biased inheritance arises from acombi- delian inheritance.The load imposedupon the population nationof asequence-specificendonuclease inserted in the (i.e.the fraction ofthereproductive effortthat is rendered middle ofits ownrecognition sequenceand the cell ’s own unproductive)is thenequal tothe frequency of homozy- recombinational repair pathway. gotes, L = qˆ2 ,andthe mean fitness of the population is 1 The simplicity ofthis mechanism suggeststhat it may minusthis, or wˆ = 1 2 e2.For example, HEGsof yeasts beopen to human artifice. The proposedconstruct is illus- canshow extreme TRD, with e < 0.99 (Jacquier &Dujon trated in figure 1, andthe essential features are asfollows. 1985; Wenzlau et al. 1989). If oneconsiders, conserva- tively, an engineeredHEG with aTRD of e = 0.9 (this (i) AHEGis engineeredto recognize andcut a sameassumption will bemade in all numerical examples sequencein themiddle ofan essentialgene, and the in this paper), thenthe equilibrium meanfitness of the HEGis insertedinto themiddle ofits ownrecog- population will be wˆ = 0.19. That is,four-fifths of zygotes nition sequence,simultaneously disrupting thegene producedwill die,and only one-fifthwill survive torepro- andprotecting thechromosome from being cut. duce.Moreover, this load will arise relatively quickly. If Naturally occurring HEGsdo notusually disruptthe theHEG is introducedinto 1% ofthe population, thenit functionof the host gene because they are associated will take only t1 ,9 0 = 12 generationsfor theload toreach with self-splicing group Iintronsor inteins 90% ofits equilibrium value. If onecan manage torelease (Chevalier &Stoddard2001), butthe engineered aninitial frequencyof only 0.01%, thenit will take 19 elementwould not have these. generations.The progression toequilibrium is shownin (ii) The target geneis chosensuch that theknockout figure 2. mutation has little phenotypic effectin thehetero- Theseresults are fairly robustto changes in thefitness zygousstate, but is severely deleteriouswhen homo- scheme.For example, if thehomozygote has someresidual zygous(i.e. the knockout is recessive). viability (i.e.the knockout is sub-lethal),then this can (iii) Finally, theHEG is underthe control of ameiosis- actually increasethe genetic load. Load is highest justat specificpromoter, sothat heterozygouszygotes thepoint at whichthe HEG can go tofixation. The results developnormally, buttransmit theHEG to a dispro- are also robustto a certain level ofheterozygote impair- portionate fraction oftheir gametes. ment(i.e. the knockout is incompletely recessive).For example, if thehomozygote is lethal andthe heterozygote This last conditionmay berelaxed, dependingupon has afitnesslevel 90% ofthe wild-type, then wˆ = 0.192 whenthe target geneis expressed:if thetarget geneis (insteadof 0.19) and t1 ,9 0 = 14 generations(instead of 12 expressedonly in larvae, or in somatic tissues,then the generations;calculations notshown). promoter canbe adult-specific, or germline-specific. If sucha constructis
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