Omar S. Akbari, Kelly D. Matzen, John M. Marshall, Haixia Huang, Catherine M. Ward, and Bruce A. Hay

Transcription

1 Current Biology Volume 23 Sulemental Information A Synthetic Gene Drive Sytem for Local Reverible Modification and Sureion of Inect Poulation Omar S Abari Kelly D Matzen John M Marhall Haixia Huang Catherine M Ward and Bruce A Hay Sulemental Inventory Sulemental Exerimental Procedure Contruction of the bae UD MEL lamid Contruction of mirna toxin Contruction of the antidote and final UD MEL contruct Trangenei and UD MEL ytem generation Embryo viability determination Poulation cage exeriment Poulation genetic modelling and fitne cot etimation Fitne cot etimation ingle-locu Fitne cot etimation two-locu Migration threhold & confinement Poulation ureion and reverion Sulemental Reference Sulemental Figure Legend Sulemental Figure Figure S Parental and rogeny genotye and urvival for two-locu and ingle-locu UD MEL Figure S2 UD MEL ingle- and two-locu ytem are redicted to how threholddeendent gene drive in the reence of a % fitne cot Figure S3 Fate of the wild-tye allele during oulation relacement with ingle- and two-locu UD MEL element with a % fitne cot

2 Sulemental Exerimental Procedure Contruction of the Bae UD MEL Plamid The Droohila alha-tubulin 3'UTR wa PCR amlified from genomic DNA uing rimer 5'-ATG CTA GCG GAT CCG GGA ATT GGG AAT TGG GCA ATA TTT AAA TAA AGA AAA ACA GTG GGG TTT-3' and 5'-CAT CAT CAT CAT CCG AAT TCC ACA GGC CGG CCA TTG GCG CGC CGC GTC ACG CCA CTT CAA CGC-3' roducing a 363b PCR roduct containing unique AcI and FeI5' retriction ite into which ecific mirna toxin could be cloned Thee rimer overla the Droohila attp lamid [] and the 3' end of the Bicaudal C romoter The Bicaudal C romoter fragment wa amlified from genomic DNA with rimer 5'-GTT GAA GTG GCG TGA CGC GGC GCG CCA ATG GCC GGC CTG TGG AAT TCG GAT GAT GAT GAT GAT-3' and 5'-TTC AAC GCA CAC TTA TTA CGT GAG CGA TCG CAT CGC ATA ATT ATA TAA TAA TAA ACT GCA TGC CGC CA-3' roducing a 293b PCR roduct Thi roduct contain a unique AiSI retriction ite at the 5'end of the Bicaudal C romoter The rimer ued to generate the fragment overla the 5' end of alha-tubulin 3' UTR and the gyy inulator The gyy inulator wa PCR amlified from genomic DNA uing rimer 5'-ATG CAG TTT ATT ATT ATA TAA TTA TGC GAT GCG ATC GCT CAC GTA ATA AGT GTG CGT TGA-3' and 5'-GAG GCG TCC AGG ATC CCA TGG GGT TCA TCT AAT GTT TAA ACA ATT GAT CGG CTA AAT GGT ATG-3' roducing a 356b PCR roduct Thi roduct contain a unique PmeI ite between the gyy inulator and the bn romoter and thee rimer overla the 5'end of the Bicaudal C romoter and the 5' end of the bn romoter The bn romoter wa PCR amlified with rimer 5'-TTT TCT TGC CAT ACC ATT TAG CCG ATC AAT TGT TTA AAC ATT AGA TGA ACC CCA TGG GAT C-3' and 5'-CGT GAC CTA CAT CGT CGA CAC TAG TGG ATC GCT AGT TAA TTA AGC CGA ATT CGT TGA CGG TTG A-3' roducing a 787b PCR roduct which carrie a unique PacI ite on the 3' end of the bn romoter for cloning in the recue fragment Thee rimer alo overla the gyy inulator and the Droohila attp lamid bacbone Thee 4 PCR roduct were urified with Qiagen Valencia CA PCR urification column and ligated uing -te recombination technology [2] into an AcI digeted Droohila attp lamid [] roducing the UD MEL -BicC-Gy- Bn-attb lamid Contruction of mirna Toxin The Droohila mirna mir6 tem-loo wa modified to target dah o-futor myd88 a decribed reviouly [3] To mae a mirna that target dah ite rimer dah--f 5'-CTT AAT CAC AGC CTT TAA TGT AGG GAA ATA TAT AAC AAT ACA CTA AGT TAA TAT ACC ATA TCT-3' and dah--r 5'- ATG TTA GGC ACT TTA GGT ACA GGG AAA TAT ATA ACA ATA AAC TAG ATA TGG TAT ATT AAC TTA G -3' were generated To target dah ite 2 rimer dah -2-f 5'- TTA AAC TTA ATC ACA GCC TTT AAT GTA ACC AGG ATG CGA ACT ATA CAC TAA GTT AAT ATA CCA TAT CTA G -3' and dah -2-r 5'- AAT GAT GTT AGG CAC TTT AGG TAC AAC CAG GAT GCG AAC TAT AAA CTA GAT ATG GTA TAT TAA CTT AG-3' were generated To target o-fut target ite rimer o-fut--f 5'- AAA CTT AAT CAC AGC CTT TAA TGT AGT TTT ATT ACA TTG ATT ACG CTA AGT TAA TAT ACC ATA TCT AG -3' and o-fut--r 5'- AAT GAT GTT AGG CAC TTT AGG TAC AGT TTT ATT ACA TTG ATT AAG CTA GAT ATG GTA TAT TAA CTT AGC G -3' were generated To target o-fut target ite 2 rimer o-fut-2-f 5'- ATC ACA GCC TTT AAT GTC AGG ATT ATC TAC TTA AAT CCT TAA GTT AAT ATA CCA TAT CTA AGT-3' and o-fut-2-r 5'- ATG ATG TTA GGC ACT TTA GGT ACC AGG ATT ATC TAC TTA AAT ACT TAG ATA TGG TAT ATT AAC TTA AGG A-3' were generated For myd88mirna

3 target ite and 2 the rimer are a decribed reviouly [3] The above air of rimary tem loo containing PCR roduct were amlified uing rimer mir65 NotI/FeI/BglII 5'- TCG GGC GGC CGC ATT TGG CCG GCC AAA GAT CTT TTA AAG TCC ACA ACT CAT CAA GGA AAA TGA AAG TCA AAG TTG GCA GCT TAC TTA AAC TTA ATC ACA GCC TTT AAT GT- 3' and mir6 3 EcoRI/AcI/BamHI 5' - TGA AGA ATT CAT TGG CGC GCC TTT GGA TCC AAA ACG GCA TGG TTA TTC GTG TGC CAA AAA AAAAAAAAA TTA AAT AAT GAT GTT AGG CAC TTT AGG TAC-3' Thee rimer add mir6 flaning equence that are thought to romote mirna roceing and everal retriction ite PCR roduct were urified with Qiagen Valencia CA PCR urification column and then digeted with aroriate retriction enzyme For dah-o-fut- and myd88- thee were NotI and BamHI and BglII and AcI for dah-2 o-fut-2 and myd88-2 Two digetion roduct each containing a ingle mirna targeting the ame gene were ligated together and cloned into the Ac5/V5-HiB huttle vector Invitrogen roducing a air of mirna deigned to ilence dah o-fut or myd88 The coy number of mirna in Ac5 wa doubled by digeting the above lamid with either NotI and BamH or BglII and AcI The roduct generated each contain 2 mirna Thee were ligated together into the Ac5 huttle vector cut with Not and Ac roducing a final vector containing two coie each of 2 mirna deigned to ilence each gene Thi rocedure wa reeated once more to roduce olycitronic 8-mer mirna toxin ecific to dah o-fut or myd88 Thee mirna were then digeted out of the Ac5 huttle vector uing FeI/AcI and ligated into UD MEL -BicC-Gy-Bn-attb reviouly digeted withfei and AcI generating UD MEL -BicC-o-fut-Gy-Bn-attb UD MEL -BicC-dah-Gy- Bn-attb and UD MEL -BicC-myd88-Gy-Bn-attb Contruction of the Antidote and Final UD MEL Contruct The O-fut coding region wa amlified from a cdna library uing rimer o-fut-anti-f 5'-ACA TTC GTA CTT CAA CCG TCA ACG AAT TCG GCT TAA TTA AAT GCA GTG GCT CAA AAT GAA GC-3' and o-fut-anti-r 5'-AGA AGT AAG GTT CCT TCA CAA AGA TCC TGC GGC CGC TTA CAG CTC CTC GTG CAC GTT TGT-3' roducing a 284b PCR roduct bearing a unique PacI retriction ite at the 5' end of the o-fut CDS and a unique Not I ite at the 3' end Thi PCR roduct overla the 3' end of the bn romoter and the 5' end of the SV4-3'UTR The SV4-3'UTR wa PCR amlified uing rimer Sv4-f 5'-TAC AAA CGT GCA CGA GGA GCT GTA AGC GGC CGC AGG ATC TTT GTG AAG GAA CCT TAC TTC-3' and Sv4-R 5'-TAC AAA CGT GCA CGA GGA GCT GTA AGC GGC CGC AGG ATC TTT GTG AAG GAA CCT TAC TTC-3' from UWG [4] roducing a 764b roduct that overla the 3'end of the o-fut CDS and the attb bacbone Thee 2 PCR roduct were ligated together uing one-te recombination a above into a PacI-digeted UD MEL -BicCdah-Gy-Bn-attb roducing final UD MEL -dah T -o-fut A Note that becaue the o-fut trancrit lac the 3' UTR reent in the endogenou o-fut trancrit it i not ilenced by mir6-o-fut- or mir6-o-fut-2 which target the native o-fut 3' UTR The dah coding region wa amlified from a cdna library uing rimer dah-anti-f- 5'-TCA ACA GCA CAT TCG TAC TTC AAC CGT CAA CGA ATT CGG CAT GCT GAG ATC GTC GGT GCC CGT-3' and dah -anti-r- 5'- GTT GCC CTG TCC AAC TTG TAA TTG GCG TCT TGA TTG AAA TGG CCT AGT TTC TCG CAG GC-3' roducing a 455b PCR roduct and dah -anti-f-2 5'- GCC TGC GAG AAA CTA GGC CAT TTC AAT CAA GAC GCC AAT TAC AAG TTG GAC AGG GCA AC-3' and dah -anti-r-2 5'- AGA CCG TGA CCT ACA TCG TCG ACA CTA GTG GAT CTC TAG CGG CCG CTC ACG TGC TGA TGC GC -3' roducing a 638b PCR roduct bearing a unique NotI ite 3' end of the dah recue The dah 3' UTR wa amlified from genomic

4 DNA with rimer dah-utr-f- 5'-CAT CAG CAC GTG AGC GGC CGC AAC GGT ACC GGA TC-3' and dah-utr-r- 5'-GAG ACC GTG ACC TAC ATC GTC GAC ACT AGT GGA TCT CTA GAG CAT TGG AAA TCT ACA AAG TTG AT-3' roducing a 374b PCR roduct that overla the dah CDS and attp bacbone Thee three PCR fragment were ligated together into PacI-digeted UD MEL -BicCo-fut-Gy-Bn-attb and UD MEL -BicC-myd88-Gy-Bn-attb roducing the final UD MEL -o-fut T - DAH A and UD MEL -myd88 T -DAH A contruct Note that becaue thi dah trancrit lac a 5' UTR reent in the endogenou dah trancrit it i not ilenced by mir6-dah- which target the dah 5' UTR mir6-dah-2 target exon 2 of the CDS in dah To render the antidote inenitive to thi mirna we recoded the nucleotide equence in dah in uch that it code for the ame amino acid equence but i not targeted by the mirna The myd88 coding region wa PCR amlified from genomic DNA uing rimer myd88-f- 5'-ATT CGT ACT TCA ACC GTC AAC GAA TTC GGC TTA ATT AAA TGC GCC CTC GAT TT GTA TGC C-3' and myd88-r- 5'-GTA AGG TTC CTT CAC AAA GAT CCT CTA GAC CGC GGC CGC TCA GCC CGG CGT CTG CAG CTT GC-3' roducing a 69b PCR roduct bearing a unique PacI ite on the 5' end of the myd88 CDS and a unique NotI ite on the 3' end of the myd88 CDS The myd88 CDS PCR roduct overla the 3' end of the bn romoter and the 5' end of the SV4 3' UTR The SV4 3' wa PCR amlified uing Sv4- M-F 5'-CAG CAG CAA GCT GCA GAC GCC GGG CTG AGC GGC CGC GGT CTA GAG GAT CTT TGT GAA GGA ACC-3' and SV4-M-R 5'-CCT ACA TCG TCG ACA CTA GTG GAT CTC TAG AGG ATC CAG ACA TGA TAA GAT ACA TTG ATG-3' roducing a 775b PCR roduct that overla the 3' end of the myd88 recue and the attp lamid Thee 2 PCR roduct were ligated together into PacI digeted UD MEL -BicC-dah-Gy-Bn-attb uing one te recombination roducing the final UD MEL -dah T -myd88 A contruct Note that becaue thi myd88 trancrit lac the 5' UTR reent in the endogenou myd88 trancrit it i not ilenced by myd88- and myd88-2 mirna toxin which are deigned to target the myd88 5'UTR Trangenei and UD MEL Sytem Generation Germline tranformation of D melanogater wa erformed by Rainbow Trangenic Flie Inc wwwrainbowgenecom Camarillo CA The trangenic line were generated by ite ecific PhiC3 integration into att docing ite on either the 2nd chromoome at cytological location 3B Bloomington fly toc # 9724 PBacy[+]-attP-3B}VK3a contruct UD MEL -o-fut T - DAH A and UD MEL -myd88 T -DAH A or the 3rd chromoome at cytological location 86E8 Bloomington fly toc # Mva-intDmZH-2A M3xP3-RFPattP'ZH-86Fa 2A3 contruct UD MEL -dah T -myd88 A UD MEL -o-fut T -DAH A UD MEL -myd88 T -DAH A and UD MEL -dah T -ofut A Each of thee line UD MEL -dah T -myd88 A at attb ite 86fa UD MEL -o-fut T -DAH A at attb ite 9724 UD MEL -o-fut- T -DAH A at attb ite 86FA UD MEL -myd88 T -DAH A at attb ite 9724 UD MEL - myd88 T -DAH A at attb ite 86FA and UD MEL -dah T -o-fut A at attb ite 86fa were maintained 25+ generation by out croing the trangenic male to WT w 8 +/+ virgin female To generate the ingle and two locu UD MEL configuration virgin female carrying one toxin-antidote combination were croed with male carrying the comlementary toxinantidote combination to roduce table line In the cae of two-locu UD MEL homozygoity for each chromoome wa achieved by carrying out ingle air croe between animal that mut be at leat tranheterozygou for both contruct for multile generation Homozygoity wa confirmed uing ingle fly PCR involving rimer deigned to amlify equence to either ide of the att inertion ite For the 2nd chromoome 9724 inertion ite rimer 9724-F 5'- ACA

5 TTT ATA TTT TCG TTT GCG ACC GA-3' and 9724-R 5'-CCC AAA AGA CTT GGC TCG GAT GCA CTG A-3' were ued For the third chromoome 86FA inertion ite we out-croed individual male to wild-tye w 8 +/+ virgin female and determined whether all offring carried the 3x3 RFP trangene aociated with thi inertion ite We alo ued PCR rimer 86fa-F 5'-ATC TGT AGG CTA GCG TAT TTA G-3' and 86fa-R 5'-GAT CCA AAA GAA TAC ATA GCA ATG CGA-3' to carry out PCR from ingle flie Thee rimer roduce an 88b PCR roduct from wild-tye DNA Embryo Viability Determination For embryo viability count 2-4 day old adult virgin female were allowed to mate with male of the relevant genotye for 2-3 day in egg collection chamber ulemented with yeat ate On the following day a 3 hr egg collection wa carried out after firt having cleared old egg from the female through a re-collection eriod on a earate late for three hr Embryo were iolated into grou of and et on an agar urface at 25 o C for hr The % urvival wa then determined by counting the number of unhatched embryo One grou of embryo er cro were cored in each exeriment and each exeriment wa carried out three time The reult reented are average from thee three exeriment Embryo urvival wa normalized with reect to the % urvival oberved in arallel exeriment carried out with the w 8 train ued for line maintenance For the embryo count for croe between heterozygou female and either heterozygou male or WT male we oberved % maternaleffect illing with no urviving embryo with a amle ize of greater then embryo for each contruct Figure 5A For Adult fly count figure 5A-bottom individual heterozygou male M/+ were mated to multile WT +/+ virgin female N=3-5 for each of the ix contruct teted and thi exeriment wa executed three time % of the rogeny from thee croe between rogeny were counted and the reult of the three exeriment were averaged together Poulation Cage Exeriment All fly exeriment were carried out at 25 o C ambient humidity in 25 ml bottle containing Lewi medium ulemented with live dry yeat Drive exeriment were teted againt a w 8 white eyed wild-tye train and fly rearing wa carried out in a light-tight dar chamber to eliminate any fitne benefit aociated with exreion of the w+ trangene G flie were collected a virgin male and female were collected concurrently and then aged for 3 day before croe were et Flie were anethetized concurrently and introduced to the bottle in a ingle grou o that no bia wa introduced from ome flie waing before other The larget ractical oulation N=5 were ued to enure good mixing a thi wa found to be imortant in the firt generation data not hown Flie were then allowed to mate and lay egg for 5 day at which oint adult were cleared from the bottle 4 day ot-mixing flie were anethetized and divided into two grou One grou wa immediately introduced into the new bottle while the other wa counted to determine genotye/henotye frequencie wild-tye and trangene-bearing were the only clae cored For exeriment in which male were releaed in both the firt and econd generation male were again collected young and aged for three day before introduction into bottle Progeny male and female from the firt generation cro were collected a virgin and young male and et iolated for three day Young tranheterozygou male from the UD MEL toc were collected and imilarly aged at the

6 ame time Subequently tranheterozygou male and a cohort of flie from the firt generation were introduced together into a new oulation cage maintaining the 6% introduction frequency reent in the firt generation Subequent generation were cored a above Poulation Genetic Modelling and Fitne Cot Etimation We ue a imle difference equation framewor to model the read of ingle and twolocu UD MEL through a randomly mating oulation In doing o we aume dicrete generation and infinite oulation ize To invetigate the confinement roertie of ingleand two-locu UD MEL we follow the framewor of Marhall and Hay [5] We conider two cenario one in which migration occur before mating mi-ma and a econd in which mating occur before migration ma-mi Detail are rovided below Fitne Cot Etimation Single-Locu In order to etimate the fitne cot of the UD MEL contruct for both ingle and two-locu ytem we aume dicrete generation random mating and infinite oulation ize There are two trangenic contruct each coniting of a maternal toxin and a zygotic antidote to the toxin on the comlementary contruct For the ingle-locu cae both of thee are reent at the ame locu We denote the trangenic allele by A and B and the null allele at thi locu by a There are ix oible genotye AA BB Aa Ba and aa and we denote the roortion of the th generation that are fruit flie having thee genotye by Aa and reectively By conidering all oible mating air taing into account that offring are unviable if they do not have the antidote for any maternal toxin oeed by their mother the genotye AA BB Aa Ba aa of embryo in the next generation are decribed by the ratio ˆ : ˆ : ˆ : ˆ : ˆ : ˆ The equation for thee ratio follow from the chematic in ulementary Figure A; however given the large number of oible mating air it i not feaible to how them here The imulation code that utilize them i available from the author uon requet The genotye frequencie in the next generation can then be calculated by accounting for the genotyeecific fitne cot ie AA BB Aa Ba aa AA BB Aa Ba aa ˆ ˆ ˆ ˆ ˆ ˆ Here Ba aa AA BB Aa Ba / W rereent the fitne cot aociated with each genotye with the genotye being lited a ubcrit W ˆ W i a normalizing term given by AA BB ˆ ˆ AA Aa Ba aa ˆ Aa ˆ Ba ˆ We invetigated a number of different fitne cot model and teted them by eeing which rovide the bet fit to the data The imlet model i one in which all trangenic have the ame fitne cot ie AA BB Aa Ba An alternative model aume that BB AA BB

7 fitne cot are additive ie AA BB and Aa Ba / 2 In both cae we alo looed into model where fitne cot varie linearly with trangenic allele frequency AA BB Aa Ba tr wt 5 wt and where it varie linearly with wild-tye frequency aa wt tr tr Here wt rereent the fitne cot of a trangenic homozygote in a nearly fully wild-tye tr oulation and rereent the fitne cot of a trangenic homozygote in a nearly fully trangenic oulation Thee model were comared according to their Aaie Information Criterion AIC value The lielihood of the data wa calculated by auming a binomial ditribution of WT and red-eyed individual and by uing the model rediction to generate exected roortion for each fitne cot and model ie by calculating the log-lielihood Ri WTi aa log WTi log log L R i R i i log AA R Here i WT and i are the number of red-eyed and WT individual at generation in exeriment i and the exected genotye frequencie deend on the fitne cot model and arameter The bet etimate of the model arameter are thoe having the highet loglielihood and the bet fitting model i that having the lowet AIC value AIC 2n 2log L where n rereent the number of model arameter The AIC value and arameter etimate for each of the fitne cot model are hown in the table below Thi table how that the betfitting model i one in which fitne cot are dominant ie trangenic homozygote and heterozygote have the ame fitne cot and the magnitude of the fitne cot deend on the frequency of wild-tye ie aa individual in the oulation BB Aa Ba Fitne Cot Model AIC wt tr Contant dominant fitne cot Contant additive fitne cot Trangenic allele frequencydeendent dominant fitne cot Trangenic allele frequencydeendent additive fitne cot Wild-tye frequency-deendent dominant fitne cot Wild-tye frequency-deendent additive fitne cot

8 Fitne Cot Etimation Two-Locu For the two-locu cae the two trangenic contruct are reent at different loci At the firt locu we denote the trangenic allele by A and the null allele by a and at the econd locu we denote the trangenic allele by B and the null allele by b Thi time there are nine oible genotye AB Ab aabb aabb and aabb and we denote the roortion of the th generation that are fruit flie having thee genotye by AB and reectively By conidering all oible mating air taing into account that offring are unviable if they do not have the antidote for any maternal toxin oeed by their mother the genotye of embryo in the next generation are decribed by the ratio AB Ab aabb aabb aabb ˆ : ˆ : ˆ : ˆ : ˆ : ˆ : ˆ : ˆ : ˆ The equation for thee ratio follow from the chematic in ulementary Figure B; however given the large number of oible mating air it i not feaible to how them here and the imulation code that utilize them i available from the author uon requet The genotye frequencie in the next generation can then be calculated by accounting for the genotye-ecific fitne cot ie AB Ab aabb aabb aabb Here Ab ˆ AB ˆ Ab ˆ ˆ ˆ ˆ aabb aabb AB Ab aabb aabb / W rereent the fitne cot aociated with each genotye with the genotye being AB W lited a ubcrit W ˆ i a normalizing term given by Ab ˆ ˆ AB Ab ˆ aabb aabb aabb aabb ˆ ˆ ˆ aabb ˆ aabb ˆ A for the ingle-locu cae we invetigated a number of different fitne cot model and teted them by eeing which rovide the bet fit to the data The imlet model i one in which all trangenic individual have the ame fitne cot ie AB Ab aabb aabb A econd model aume that each trangenic allele ha the ame fitne cot ie AB Ab 2 and aabb aabb ; and a third model aume that the fitne cot of each allele are additive ie AB 2 Ab 3 / 2 aabb and aabb / 2 In each cae we alo looed into model where fitne cot varie linearly with wild-tye frequency aabb wt tr tr and where it varie with trangenic allele frequency AA Aa BB Bb 5 5 tr wt wt 2 where aabb ˆ aabb ˆ aabb ˆ

9 Here wt AA Aa BB Bb AB AB Ab Ab aabb aabb rereent the fitne cot of a trangenic homozygote at both loci in a nearly fully tr wild-tye oulation and rereent the fitne cot of a trangenic homozygote at both loci in an almot fully trangenic oulation Thee model were comared according to their Aaie Information Criterion AIC value A for the ingle-locu cae the lielihood of the data wa calculated by auming a binomial ditribution of WT and red-eyed individual and by uing the model rediction to generate exected roortion for each fitne cot and model ie by calculating the loglielihood Ri WTi aabb log WTi log Ri log L AB Ab i R log i R Here i WT and i are the number of red-eyed and WT individual at generation in exeriment i and the exected genotye frequencie deend on the fitne cot model and arameter The bet etimate of the model arameter are thoe having the highet loglielihood and the bet fitting model i that having the lowet AIC value The AIC value and arameter etimate for each of the fitne cot model are hown in the table below Interetingly the bet-fitting model for the two-locu ytem i the ame a for the ingle-locu ytem In thi model fitne cot are dominant uch that all trangenic individual uffer the ame fitne cot and the magnitude of the fitne cot deend on the frequency of wild-tye ie aabb individual in the oulation aabb aabb Fitne Cot Model AIC wt tr Contant dominant fitne cot Contant dominant allelic fitne cot Contant additive allelic fitne cot Trangenic allele frequency-deendent dominant fitne cot Trangenic allele frequency-deendent dominant allelic fitne cot Trangenic allele frequency-deendent additive allelic fitne cot

10 To ummarize the bet-fit model Table S for ingle-locu UD MEL i one in which fitne cot are dominant ie trangenic homozygote and heterozygote have the ame fitne cot and the magnitude of the fitne cot deend on the frequency of wild-tye ie aa individual in the oulation We calculated 95% confidence interval for the arameter ued in thi model uing a Marov Chain Monte Carlo amling rocedure Interetingly reult ugget that trangenic exerience a fitne benefit in a nearly fully wild-tye oulation of 242% 95% confidence interval: 25%-276% and a trangenic fitne cot in a nearly fully trangenic oulation of 276% 95% confidence interval: 265%-286% The bet-fit model for the twolocu ytem ha imilar overall characteritic Table S2 though the calculated fitne cot are different The bai for thee effect remain to be exlored Migration Threhold and Confinement We firt invetigate the confinement roertie of ingle-locu UD MEL following the framewor of Marhall and Hay [5] in which migration occur before mating We conider a two-oulation model in which the mating ool in both oulation i made u of individual from oulation and 2 The roortion of the th generation that are individual having the genotye AA AA BB Aa Ba aa BB Aa Ba and aa in oulation are denoted by and reectively with correonding ymbol alying to oulation 2 For a migration rate of in both direction we mae the following ubtitution into the equation decribed earlier for UD MEL dynamic in oulation Iterating thee equation confirm that ingle-locu UD MEL i confineable to a artially-iolated oulation and that rather than reading into neighboring oulation at high migration the ytem i actually eliminated from both oulation imilar to the cae for ingle-locu engineered UD We alied the ame modeling framewor to determine the migration and confinement roertie of the two-locu UD MEL ytem In the cae where mating occur before migration the analyi roceed imilarly We conider a two-oulation model in which mating occur in oulation and 2 earately following which each oulation exchange a fraction of it oulation with the other The roortion of the th generation that are individual having the genotye AA BB Aa Ba and aa in oulation are denoted by and reectively with correonding ymbol alying to oulation 2 Iterating thee equation confirm that inglelocu UD MEL i confineable to a artially-iolated oulation and that rather than reading into neighboring oulation at high migration rate the ytem i actually eliminated from both oulation imilar to the cae for ingle-locu engineered UD A above thi ame modeling framewor wa alied to determine the migration and confinement roertie of the twolocu UD MEL ytem AA BB Aa Ba aa

11 Poulation Sureion and Reverion We invetigate the oibility of ureing and reverting UD MEL -relaced oulation uing a tochatic oulation model analogou to the oulation frequency model reviouly decribed A tochatic model wa choen becaue it can accommodate the mall oulation reulting from oulation ureion Denity-deendence i an imortant conideration becaue at low oulation ize larval cometition i reduced and a ingle female can roduce more offring than urvive to adulthood We adat a general moquito oulation biology model [67] modified to run in dicrete time with unit of one generation In thi model the number of adult female in the oulation at generation i given by N fn F L Here f f L P rereent the roortion of embryo that are viable after the toxin-antidote effect of the UD MEL contruct are taen into account f rereent the average number of female egg laid by an adult female in her lifetime rereent the roortion of egg that urvive L the egg life tage rereent the roortion of larvae that urvive the larval life tage in the F L abence of denity-deendence rereent the roortion of larvae that urvive the effect of denity-deendent mortality denity-deendence i aumed to act at the larval tage and P rereent the roortion of uae that urvive the ual life tage The denitydeendent function i given by Here Finally L F L L rereent the number of larvae of both exe at generation Thi i given by L 2 fn f i a term that ecifie the trength of denity-deendence and for a oulation with an adult carrying caacity of K i given by fk f L P N 2N The total oulation ize i given by f Stochatic effect are incororated by amling the number of individual having each genotye from a Poion ditribution with a mean equal to the exected number of individual having thi genotye For the ingle-locu ytem ureion of the UD MEL -relaced oulation can then be achieved by conecutive releae of wild-tye male and oulation reverion can be achieved by releaing mall number of wild-tye male and female into the ureed oulation o that wild-tye individual exceed the required threhold for re-colonization For the two-locu ytem oulation ureion can t be achieved through the releae of wild-tye male; however three conecutive releae of 5 wild-tye male and 5 wild-tye female are ufficient for wild-tye to exceed the required threhold for reverion to a fully wild-tye oulation

12 Parameter are decribed in the following table and code i available from the author uon requet Symbol Parameter Value Reference f Average number of female egg a female adult lay in her lifetime 3 [8] Probability of urviving the egg life tage 83 [9] L Probability of urviving the larval life tage no 76 [9] denity deendence P Probability of urviving the ual life tage 83 [9] K Adult oulation carrying caacity Sulemental Reference Fih MP Groth AC Calo MP and Nue R 27 Creating trangenic Droohila by microinjecting the ite-ecific hic3 integrae mrna and a trangene-containing donor lamid Nature rotocol Gibon DG Young L Chuang RY Venter JC Hutchion CA 3rd and Smith HO 29 Enzymatic aembly of DNA molecule u to everal hundred ilobae Nature method Chen CH Huang H Ward CM Su JT Schaeffer LV Guo M and Hay BA 27 A ynthetic maternal-effect elfih genetic element drive oulation relacement in Droohila Science Abari OS Oliver D Eyer K and Pai CY 29 An Entry/Gateway cloning ytem for general exreion of gene with molecular tag in Droohila melanogater BMC Cell Biol 8 5 Marhall JM and Hay BA 22 Confinement of gene drive ytem to local oulation: a comarative analyi Journal of Theoretical Biology Hancoc P A and Godfray H C J 27 Alication of the lumed age-cla technique to tudying the dynamic of malaria-moquito-human interaction Malaria Journal Deredec A Godfray H C J and Burt A 2 Requirement for effective malaria control with homing endonucleae gene Proc Natl Acad Sci USA 8 E874-E88 8 Molineaux L and Gramiccia G 27 The Gari Project: Reearch on the Eidemiology and COntrol of Malaria in the Sudan Savanna of Wet Africa World Health Organization Geneva 9 Deinay J M O Mbogo C M Killeen G Knol B Beier J Carlon J Duhoff J Billingley P Mwambi H Githure J Toure A M and McKenzie F E 24 A imulation model of African Anohele ecology and oulation dynamic for the analyi of malaria tranmiion Malaria Journal 3 29

13 Sulemental Figure Legend Figure S Parental and Progeny Genotye and Survival for Two-Locu and Single-Locu UD MEL Punnet quare for ingle-locu A and two-locu B UD MEL indicating all oible arental and offring genotye Progeny exected to erih are indicated in in Figure S2 UD MEL Single- and Two-Locu Sytem Are Predicted to Show Threhold-Deendent Gene Drive in the Preence of a % Fitne Cot The threhold frequency above which a UD MEL drive ytem read into a oulation and below which it i eliminated from the oulation when each element carrie a % fitne cot wa calculated uing a determinitic model and grahed a in Figure 2 Fitne cot are additive Thu for the two-locu ytem there i a % cot er locu when homozygou at that locu and a 5% cot when heterozygou Releae threhold are calculated for two ingle locu cenario: a ingle all-male releae of tranheterozygote A and two all male releae of tranheterozygote in the firt and econd generation B For X-autoome two-locu UD MEL C and autoome-autoome two-locu UD MEL D ingle releae of doubly homozygou male are illutrated Introduction frequencie/trangene frequencie rereent the fraction of individual in the total oulation carrying at leat one UD MEL contruct The ymbol refer to fitne cot Figure S3 Fate of the Wild-Tye Allele during Poulation Relacement with Single- and Two- Locu UD MEL Element with a % Fitne Cot Panel are a in Figure S2 with the Y axi indicating the wild-tye allele frequency

14 A A/B Female +/+ A/B A/B A/A B/B A/A A/B A/B B/B Male A/A A/B A/B B/B A/A +/+ A/B +/+ A/B +/+ B/B +/+ +/+ +/+ +/+ +/+ +/+ +/+ SIngle Locu UD MEL 6 dihybrid unnet Square B Female Male A/A ; B/B ; B/B +/+ ; B/B A/A ; A/A ; +/+ +/+ ; ; +/+ +/+ ; +/+ A/A ; B/B ; B/B +/+ ; B/B A/A ; A/A ; +/+ +/+ ; ; +/+ +/+ ; +/+ A/A ; B/B A/A ; B/B ; B/B A/A ; B/B ; B/B A/A ; A/A ; B/B ; B/B A/A ; ; B/B A/A ; A/A ; A/A ; B/B ; B/B ; B/B A/A ; B/B A/A ; A/A ; ; B/B +/+ ; B/B A/A ; B/B A/A ; ; B/B ; B/B A/A ; B/B +/+ ; B/B +/+ ; B/B ; B/B +/+ ; +/+ ; B/B +/+ ; A/A ; +/+ ; +/+ ; ; B/B +/+ ; B/B ; B/B ; B/B +/+ ; B/B +/+ ; +/+ ; B/B ; B/B +/+ ; +/+ ; B/B +/+ ; +/+ ; A/A ; B/B A/A ; B/B ; B/B ; B/B A/A ; B/B A/A ; A/A ; B/B ; B/B A/A ; A/A ; +/+ A/A ; A/A ; A/A ; A/A ; A/A ; +/+ ; B/B ; +/+ A/A ; +/+ ; +/+ ; +/+ ; +/+ ; +/+ A/A ; A/A ; A/A ; A/A ; +/+ A/A ; +/+ A/A ; A/A ; +/+ ; +/+ A/A ; +/+ ; +/+ ; +/+ ; +/+ ; B/B ; B/B +/+ ; B/B +/+ ; B/B +/+ ; B/B ; B/B +/+ ; B/B ; +/+ +/+ ; +/+ ; +/+ ; +/+ ; +/+ ; ; B/B ; +/+ ; +/+ +/+ ; +/+ ; +/+ +/+ ; +/+ +/+ ; +/+ ; +/+ +/+ ; +/+ A/A ; B/B A/A ; B/B A/A ; ; B/B A/A ; B/B ; B/B A/A ; ; B/B +/+ ; B/B A/A ; B/B ; +/+ ; B/B A/A ; A/A ; +/+ A/A ; +/+ ; B/B ; +/+ ; B/B A/A ; +/+ ; A/A ; +/+ +/+ ; ; +/+ A/A ; +/+ ; B/B +/+ ; A/A ; +/+ ; +/+ ; B/B +/+ ; +/+ +/+ ; +/+ ; +/+ +/+ ; B/B +/+ ; A/A ; +/+ ; +/+ ; +/+ +/+ ; +/+ +/+ ; +/+ ; +/+ A/A ; A/A ; A/A ; +/+ A/A ; +/+ ; +/+ A/A ; A/A ; +/+ ; +/+ ; +/+ ; +/+ A/A ; +/+ ; +/+ ; ; +/+ ; +/+ +/+ ; +/+ ; +/+ +/+ ; +/+ ; +/+ A/A ; +/+ +/+ ; +/+ +/+ ; +/+ +/+ ; ; +/+ ; +/+ +/+ ; ; +/+ +/+ ; +/+ +/+ ; ; +/+ +/+ ; +/+ +/+ ; +/+ ; +/+ +/+ ; +/+ 2 Locu UD MEL 8 dihybrid unnet Square

15 Trangenic frequency % C Trangenic frequency % A Single locu UD MEL = % male releae Releae at 9% Releae at 84% Releae at 83% 2 3 Generation X autoome two locu UD MEL = % male releae Releae at 5% Releae at 49% Releae at 48% 2 3 Generation Trangenic frequency % Trangenic frequency % B D Single locu UD MEL = % male releae over 2 gen Releae at 6% Releae at 5% Releae at 49% 2 3 Generation Autoome autoome two locu UD MEL = % male releae Releae at 4% Releae at 34% Releae at 33% 2 3 Generation

16 A Single locu UD MEL = % male releae B Single locu UD MEL = % male releae over 2 gen Wild tye frequency % C Releae at 9% Releae at 84% Releae at 83% 2 3 Generation X autoome two locu UD MEL = % male releae Wild tye frequency % D Releae at 6% Releae at 5% Releae at 49% 2 3 Generation Autoome autoome two locu UD MEL = % male releae Wild tye frequency % Releae at 5% Releae at 49% Releae at 48% Wild tye frequency % Releae at 4% Releae at 34% Releae at 33% 2 3 Generation 2 3 Generation