Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2

The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

For both mutations, a single guide RNA (gRNA) and a 140 nt single-stranded oligodeoxynucleotide (ssODN) repair template were designed to introduce the amino acid exchanges corresponding to the Dmel ts1 or ts2 mutations (ts1: 158 Ala > Val, ts2: 188 Pro > Ser), to create temperature-sensitive versions of the CcTRA2 protein. The repair template ssODN_tra2_ts1 differs from the wild-type tra2 ORF sequence by two bases, a C > T transition at position 473 of the CDS to introduce the ts1 SNP and the silent mutation 477 G > A that removes the PAM sequence to prevent re-editing. ssODN_tra2_ts2 differs by one base introducing the ts2 SNP (CDS: 562 C > T) (Fig. 1b).
Preliminary gRNA tests to confirm editing capability of tra2 ts positions. To assess the functionality of the tra2 ts1 and tra2 ts2 gRNAs, each was injected complexed with Cas9 protein and either without (non-homologous end joining, NHEJ, knock-out) or with repair template (homology-directed repair, HDR, knock-in). G 0 survivors of these injections were reared at 26 °C. 327 Egypt II wild-type (EgII WT) embryos were injected for tra2_ts1 knock-out. Ten reached adult stage (six males, four females) (Table 1a). One male was fertile. The ts1 injection with repair template (290 EgII embryos) yielded four viable but infertile adults (two males, two females), and three adults got stuck in the puparium while eclosing and died (two males, one female) ( Table 1a). None of the ts1 G 0 adults showed external phenotypic abnormalities. To check for editing activity of Scientific Reports | (2020) 10:18611 | https://doi.org/10.1038/s41598-020-75572-x www.nature.com/scientificreports/ gRNA_tra2_ts1, the tra2 genotype of four randomly chosen G 0 flies (two from each injection) was analysed by subcloning the tra2-specific PCR products. One of two knock-out injected G 0 flies showed a 1 bp deletion in one of five sequenced clones. One of the knock-in injected G 0 flies showed two independent events within five sequenced clones, the tra2 ts1 HDR genotype or a 6 bp deletion ( Supplementary Fig. S1a). The tra2 ts2 gRNA knock-out injection yielded six adult males from 367 injected EgII embryos (Table 1b), three of them were fertile. Additionally, three G 0 flies stuck in the puparium did not survive (two males, one intersex IS1-KO). The tra2 ts2 knock-in mix was injected into 244 EgII embryos (Table 1b). Eight developed to adults (six males, two intersex: IS1, IS2), and four died during eclosing (one male, three intersex: IS3-6). Intersex flies showed varying degrees of phenotypically male and female characteristics (genital terminalia apparatus and bristles) (Fig. 2a), and were sterile. In contrast, all six G 0 males were fertile. The genotype of six ts2 G 0 flies from the knock-out (males M5, M6, and intersex IS1-KO) and knock-in injection (IS1, IS4, IS5) was analysed. All showed NHEJ events ranging from 33 bp deletions to 4 bp insertions ( Supplementary Fig. S1b). G 1 offspring from both injections was not analysed.
These experiments confirmed the editing activity of the ts1 and ts2 gRNAs. The lack of fertile G 0 in the ts1 injections and the complete lack of females and appearance of intersexes in the ts2 injections, however, indicated that 26 °C is a restrictive temperature for the Cctra2 ts mutations.  capitata and D. melanogaster TRA2. Shown are the RNA recognition motif (RRM, black), two ribonucleoprotein identifier sequences (RNP motifs, grey), the linker region (blue), and the position of the tra2 ts1 and tra2 ts2 mutations (red). Consensus is shown in black, amino acids with similar characteristics in grey. (b) Overview of Cctra2 gene structure (tra2 exon structure, light grey: CDS, dark grey: UTR), primers used for genotyping (P1500/P1401) or for genomic positive control PCRs (P1500/P1532 and P1500/P1401), position of single guide RNAs (blue arrows) and mutations mediated by the HDR repair templates (ssODN). PAM sequences are marked in light yellow, position of SNPs introduced by HDR are shown and marked either in blue (silent mutation) or in red (functional mutations). Resulting amino acid exchanges are indicated.  S3c). Flies with intersex phenotype showed apparently normal ovaries but no spermathecae (IS6), hypertrophic testes (IS8), miniaturized testes (IS13), or no identifiable reproductive organs (IS7, IS11, IS12; Fig. 2b).

Evaluation
To assess the karyotype of all 13 G 0 flies, PCR on Y-chromosome-specific repetitive elements was performed, whereby absence of a PCR signal implies a XX-karyotype. None of the intersex phenotype G 0 flies was positive for the Y-chromosome-specific PCR (Fig. 2c), indicating that all XX (female) karyotype G 0 embryos were transformed to intersex flies. The absence of phenotypically female G 0 in all three tra2 ts2 injections indicates a high efficiency of gRNA_tra2_ts2 and the importance of the targeted position for proper TRA2 function in female sex development.
The tra2 genotype of the G 1 flies was analysed via non-lethal genotyping. For family M8, ten of twelve analysed G 1 (83%) were heterozygous for the knock-in genotype (tra2 ts2 ), and two (17%) carried NHEJ events. Table 1. Summary of injections for targeted Cctra2 knock-out or knock-in mutations. Shown is the mutation target tra2 ts1 (a) and tra2 ts2 (b), the strategy (knock-out (KO) or knock-in (KI)), the rearing temperature, the number of injected embryos and surviving G 0 larvae, pupae and adults, the larval and adult hatch rate, and the number, phenotypic sex and fertility of viable G 0 adults. Number of fertile flies for the tra2 ts1 KI injection at 19 °C could not be exactly assessed, as only twelve flies were backcrossed individually and remaining 21 flies were backcrossed in three groups.
Inbreeding of the ts2 mutation at 19 °C does not produce phenotypic females homozygous for tra2 ts2 . Heterozygous tra2 ts2 mutant G 1 flies were either inbred or backcrossed to EgII to ensure the propagation of the line if inbreeding should turn out to be sterile. Details on crosses, egg collection numbers, and temperature profiles are shown in Supplementary Tables S1, S3, and Fig. S2. Inbreeding of heterozygous M8 offspring produced 121 G 2 flies with a 1:2 female to male ratio (Supplementary Table S3). 27 of 78 phenotypic G 2 males were homozygous for the ts2 mutation (tra2 ts2|ts2 ), 38 were heterozygous (tra2 ts2|WT ), and 13 were WT (tra2 WT|WT , Fig. 3a). In contrast, none of the 38 phenotypic females were homozygous for tra2 ts2 , 24 were heterozygous, and 14 had two WT tra2 alleles (Fig. 3a). Inter se crosses of M11 offspring resulted in a similar phenotypic female to male ratio as M8 inbreeding (26 and 42, respectively). Non-lethal genotyping showed that also M11 inbreeding produced phenotypic tra2 ts2 -homozygous males (21%), but no phenotypic females with two tra2 ts2 alleles (Fig. 3a). Backcross of tra2 ts2 heterozygous M11 offspring produced a 1:1 phenotypic sex ratio (Supplementary Table S3), which was not further analysed molecularly. using the same DNA samples as in the Y-specific PCR, to exclude lack of PCR product due to DNA quality. Individuals lacking a signal in the Y-chromosome-specific PCRs but not in the genomic control PCR are marked in bold letters to indicate the XX-karyotype. M11ib_m29 was excluded from the analysis, due to low DNA quality. One phenotypic male (WT m) and female (WT f) from family M8 with WT tra2 genotype are shown as controls. Displayed are cropped parts from different gels. Uncropped versions of the gels are provided in the supplement (Supplementary Fig. S5a  of phenotypic females homozygous for the tra2 ts2 mutation in G 2 implied that XX embryos homozygous for tra2 ts2 are either not viable or transformed into phenotypic males at 19 °C. Y-specific primers were used to assess the karyotype of 35 G 2 tra2 ts2 -homozygous and 60 heterozygous male G 2 flies by PCR. In family M8, nine of 27 phenotypic males homozygous for tra2 ts2 showed a signal in the control genomic PCR but not in the Y-chromosome-specific PCR, confirming the transformation of tra2 ts2homozygous XX flies into phenotypic males. This also applied to two out of eight phenotypic males in family M11 (Fig. 3b). For one M11 offspring, M11ib_m29, no statement can be made as the control PCR failed to produce a signal. In contrast, all tra2 ts2heterozygous males were positive for the Y-chromosome-specific PCR ( Supplementary Fig. 6), excluding sex conversion as reason for the male-biased sex ratio in the G 2 heterozygotes. Dissection of six XX-and four XY-karyotype males homozygous for tra2 ts2 , and two XY tra2 ts2 -heterozygous males (all G 2 ) showed that all tra2 ts2 -homozygous males (XX and XY) had apparently normal or slightly decolorized testes. The two tra2 ts2 -heterozygous males, in contrast, showed severely decolorized testes (Fig. 3c). In addition, across the G 2 offspring of both families, M8 and M11, 81.8% of the tra2 ts2 homozygous XX males, 4.3% of the tra2 ts2 homozygous XY males, and 16.6% of the tra2 ts2 heterozygous XY males were not capable to coil and store their distiphallus (Fig. 3c). This phenotype was also observed in random samples of WT flies of different ages; while its penetrance in WT is higher at 19 °C (24.8%, n = 161) than at 26 °C (6.9%, n = 174), it is still markedly lower than observed in the tra2 ts2 homozygous XX males (81.8%, n = 11) and might, therefore, be also an effect of the ts2 mutation.
Rearing at lower temperature leads to low fertility rates. Based on the karyotyping experiments, 19 °C still is a restrictive temperature for the ts2 mutation in Cctra2, contrary to D. suzukii tra2 ts2 where 20 °C was permissive 38 . Data from D. melanogaster suggests 16 °C as permissive temperature 30,31 . However, medflies do not breed at such low temperatures, as the small-scale fertility tests at 16 °C had shown. To attain a permissive temperature for the medfly tra2 ts2 mutation that does not affect breeding, the temperature was lowered to 18.5 °C, the mating threshold temperature 41 , for G 2 crossing and egg laying (Supplementary Table S1, Fig. S2c). ts2-homozygous XX and XY G 2 males were backcrossed to EgII females individually (13 crosses) or in groups (two crosses). ts2-heterozygous males and females were inbred (three crosses) or backcrossed (one group). Overall, during 13 days and 81 egg collections, more than 8,000 eggs were collected from these 19 crosses (Supplementary Table S4). A total of five larvae hatched from two egg collections of homozygous tra2 ts2 XY male group-backcrosses, and only one survived to adulthood (M11ib_m1-het, Supplementary Table S4). Noteworthy, due to technical restrictions the temperature could not be kept constantly at 18.5 °C during the experiment, and these larvae hatched from a late egg collection (383 h; Supplementary Fig. S2c), prior to which the temperature had been above 18.5 °C for about two days. The male (G 3 ) was crossed to 40 EgII females but did not reproduce. Therefore, maintaining the ts2 mutant strain by lowering the temperature to a permissive range was not possible.

Discussion
CRISPR/Cas9-HDR gene editing was used to create temperature-sensitive mutations in the C. capitata sex-determination gene transformer-2, equivalent to the two chemically induced point mutations in D. melanogaster 30,31 . The D. melanogaster tra2 ts temperature-dependent sex-conversion phenotype promises great advantages for creating male-only populations needed for SIT programs, as it doubles the amount of male offspring per parental egg capacity, and only heat is needed for induction. Some countries do not regulate the use of organisms carrying CRISPR-induced SNPs as they could have also occurred by natural mutagenesis and selective breeding 40,42 . Hence, only the tra ts SNPs, but no exogenous DNA was inserted, to help facilitate a potential field release of Cctra2 ts strains. This was possible due to the high mutagenesis rate achieved with our previously published CRISPR/ Cas9-HDR protocol 39 , which we now successfully applied for the first time without using a visible phenotype.
The injections aiming at creating the tra2 ts1 allele did not result in any mutant G 1 offspring at 19 °C, despite promising prerequisites; ts1 gRNA and ssODN were functional in the preliminary tests at 26 °C, and the high number of G 0 adult survivors in the 19 °C injection increased the chance to obtain mutant offspring. Moreover, G 0 flies showed deformities of internal reproductive organs (Supplementary Fig. 3b). It can't be excluded, however, that these are the result of physical damage to the embryo caused by the injection. Possible reasons for the poor efficiency of the ts1 knock-in could be the low on-target activity score of the ts1 gRNA (0.045), or a stronger phenotypic impact of the tra2 ts1 mutation compared to tra2 ts2 as observed in D. melanogaster 30 , which could reduce the chance to obtain viable ts1 mutant flies. Testing of other ts1 gRNAs could shed more light on possible reasons for the failure to create a stable ts1 line; but considering the decreased viability in D. melanogaster and the permissive temperature issues in medfly, these experiments have little prospect for success.
In contrast, the tra2 ts2 mutation could be introduced with high efficiency, detectable already from the absence of phenotypic females and the appearance of intersexes in G 0 , in the frequency of HDR-positive fertile G 0 (100% at 19 °C), as well as in the high penetrance of the mutant genotype within their G 1 offspring (83% and 75% knockin for family M8 and M11, respectively). This matches the higher on-target activity score of the ts2 gRNA (0.140).
The observed overall higher survival rate of injected G 0 at 19 °C compared to 26 °C might be the result of a lower Cas9 editing activity 43 and a potentially associated off-target rate, but could also be connected to the reduced speed of embryonic development allowing more time for repair mechanisms to fix injection-induced damage to the embryo 44 , which is unrelated to Cas9 editing. Extensive comparative injections would be needed to answer this question.
The lack of phenotypic females homozygous for tra2 ts2 and the conversion of XX embryos into phenotypic males at 19 °C suggests that this is still a restrictive temperature for the Cctra2 ts2 mutation, which does not allow correct protein folding, and indicates the importance of this position in the highly conserved TRA2 linker region Scientific Reports | (2020) 10:18611 | https://doi.org/10.1038/s41598-020-75572-x www.nature.com/scientificreports/ for correct protein conformation. This observation is in line with the results obtained for the D. melanogaster tra2 ts2 mutation, where the temperature had to be lowered to 16 °C to generate fertile males and females, while 18 °C produced sterile males and females, and 29 °C resulted in sterile males and pseudomale-like intersexes 30 . A further reduction of the temperature to an average of 18.3 °C, however, resulted in a loss of the strain due to mainly unviable eggs deposited by the G 2 generation. This was not unexpected since our small-scale tests with WT at 18 °C and 16 °C produced very little or no viable offspring, respectively. Furthermore, some males were not capable of coiling and storing their distiphallus. While this phenotype was also observed in WT males at low temperatures, it seems to be enhanced by the tra2 mutant allele. However, the numbers are too small for a robust statement. Fertility and mating behaviour of this phenotype have not been assessed. While the mean survivorship of medfly egg and larval stages at 15, 20, 25 and 30 °C has been reported to not differ significantly 21 , and the described threshold for ovarian maturation with 8.1 °C to 16.6 °C 21,41,45 is also below the tested Cctra2 ts2 permissive temperature of 18.3 °C, Prokopy and Hendrichs 41 showed that 18.5 °C is the temperature threshold for mating in medfly. During the cross of tra2 ts2 G 2 flies, temperatures were above the threshold mainly during the first days (1-72 h) and last days (337-517 h) of the crossing (Supplementary  Table S1, Fig. S2c). As ovarian maturation takes up to 10 days at this temperature, and crosses have been set up with 3-5 d old flies, no successful mating could have been achieved during the first period above 18.5 °C. During the main egg collection period (72-336 h), temperature was mainly below 18.5 °C (Supplementary Fig. S2c). The successful mating appeared within the second period of exceeded temperature. A possible explanation for the loss of the tra2 ts2 strain therefore is that the low temperatures prevented mating and eggs have not been fertilized until temperatures had exceeded 18.5 °C for at least 2 d. On the other hand, control crosses of EgII flies managed to produce a small amount of offspring at temperatures mainly lower than or equal to 18.5 °C (2,796 collected eggs, 16 larvae, 8 adults; Supplementary Fig. S2c), showing that low mating activity is taking place at or below the threshold. Therefore, it is possible that the ts2 mutation, even in the heterozygous state, affects the fertility of the flies at temperatures lower than 18.5 °C. However, as numbers are very small, no robust statement is possible. Overall, using the EgII background for the tra2 ts experiments, it could not be determined if the permissive temperature for the medfly tra2 ts2 mutation is lower than 18.5 °C or if the ts2 mutant phenotype in medfly is not temperature-dependent at all.
As strains with different genetic backgrounds can have markedly different sensitivities for elevated or low temperatures due to adaptation mechanisms, using another medfly WT background might allow to investigate lower permissive temperatures for ts2. It might also be possible to induce cold acclimation in a WT strain by successively reducing the rearing temperature over several generations before generating the tra2 ts2 mutation. This strategy would fail, however, if there is no acclimation with respect to the mating threshold, as shown for B. tryoni 46 .
Moreover, with regard to the use of the tra2 ts2 mutation for medfly sexing in a mass-rearing facility, the presumably low (< 18.5 °C) permissive temperature of the medfly ts2 mutation would be problematic, as temperature and development time show a linear relation. At 19 °C, for example, the development from egg to adults takes about 32.7 d plus 9 d for ovarian maturation, compared to 17.4 d plus 5.3 d at 26 °C 21 . The even longer development times at < 18 °C would thus be problematic for the production scale and the cost-effectiveness of a massrearing program and investigations into lower temperatures would thus certainly not be relevant for insect pest control applications in medfly.
In conclusion, we demonstrated the successful creation of the D. melanogaster tra2 ts2 point mutation in C. capitata via markerless CRISPR/Cas9-HDR gene editing and the importance of the respective amino acid for the correct function of TRA2 in the female sex-determination. The previously shown high HDR efficiency in medfly using a ssODN repair template to convert the marker gene eGFP (enhanced green fluorescent protein) into BFP (blue), could be confirmed in this study, where we achieved 100% knock-in efficiency (2 out of 2 fertile G 0 ) compared to 86% (6 out of 7 fertile G 0 ) in the previous study 39 . Also, the high penetrance of mutant offspring within the G 1 with 75-83% in this study is similar compared to 90% in the previous one. It was not possible, however, to identify a permissive temperature at which the tra2 ts2 mutation does not affect female development, as it would be located below the mating threshold of medfly. Therefore, it could not be determined if we hadn't reached the permissive temperature yet, or if the tra2 ts2 phenotype in medfly, in contrast to Drosophila, is not temperature dependent. Based on the data presented here, a medfly sexing strain built solely on tra2 ts2 would be unsuitable for an SIT program and mass-rearing, either because the rearing would be too slow to be productive on a large scale, or because the sex conversion could not be switched off for strain maintenance. Other possibilities to create a sex-conversion system in medfly could be to target other sex-determination genes, like transformer 27,29,47 , or to force (over)expression of the maleness factor MoY, which induces masculinization in XX embryos 48 . However, conditionality would need to be engineered for both options.

Material and methods
Rearing conditions. Ceratitis capitata wild-type Egypt-II (EgII) flies were received from the FAO/IAEA Agriculture and Biotechnology Laboratory, Austria, and kept at 26 °C, 48% RH and 14/10 h light/dark cycle. For fertility tests, freshly eclosed EgII adult flies were transferred from 26 °C to 19.5 °C, 60% RH, 24 h light or 16 or 18 °C, 46-48% RH, 24 h light, where egg collections and subsequent rearing took place. tra2 ts mutants were kept at 19 °C or 18.5 °C, 60% RH, and 24 h light. Temperature and humidity were measured every five minutes of the experiment using an EL-USB-2 data logger (Lascar electronics, measurement precision for temperature ± 1 °C, for humidity ± 3%). Readout of the data logger showed that during the rearing of the tra2 ts2 mutants, short-term variations of the temperature (+ 3 °C/− 1 °C) occurred (see Supplementary Table S1 and Fig. S2). These could not be avoided due to technical restrictions of the experimental setup. Furthermore, the targeted temperature (19 °C) was once exceeded for 3.5 h up to a temperature of 25 °C during an outage of the air conditioning sys- and assessment of potential off-target effects was performed using the C. capitata genome version Ccap 2.1 (GCF_000347755.3, NCBI) 49 and the Software Package Geneious Prime 50 . On-target activity score was 0.045 for gRNA_tra2_ts1, and 0.140 for gRNA_tra2_ts2 (scores are between 0 and 1; 1 = highest expected activity 50 ). Both gRNAs showed zero off-targets sites in the medfly genome. gRNA synthesis, in vitro transcription and purification was performed as described before 39 , using primers P_1439 (GAA ATT AAT ACG ACT CAC TAT AGG TGA  TGA TAT AGC TGA TGC TAG TTT TAG AGC TAG AAA TAGC) 39,51 . For knock-in experiments, 200 ng/µl ssODN_tra2_ts1 or ssODN_tra2_ts2 were added to the mix. The mixes were freshly prepared on ice, incubated at 37 °C for 10 min to allow pre-assembly of gRNA-Cas9 ribonucleoprotein complexes and stored on ice prior to injections. For microinjection of WT C. capitata embryos, eggs were collected over a 30-50 min period, prepared for injection and handled afterwards as previously described 39 . Injections were performed using siliconized quartz glass needles (Q100-70-7.5; LOT171381; Science Products, Hofheim, Germany), drawn out on a Sutter P-2000 laser-based micropipette puller. Injection equipment consisted of a manual micromanipulator (MN-151, Narishige), an Eppendorf FemtoJet 4i microinjector, and an Olympus SZX12-TTR microscope (SDF PLAPO 1xPF objective). Injection survivors were numbered successively across ts1 injections and ts2 injections, respectively.
Crossing strategies and dissection of internal reproductive organs. Crossing of G 0 : Each G 0 adult injection survivor was individually crossed to three EgII WT males or virgin females, except for the 19 °C ts1 knock-in injection. Here, six males and six females were individually backcrossed, the remaining flies were group-backcrossed (five G 0 males to 15 females, ten G 0 females to ten WT males, and six G 0 females to nine WT males). Eggs were collected three to five times, with an interval of one to two days. For the 19 °C knock-in experiments, G 1 and G 2 flies (if applicable) were kept individually until their genotype was assessed via nonlethal genotyping.
Crossing of tra2 ts2 G 1 : males and females heterozygous for tra2 ts2 were inbred. Additionally, heterozygous males were backcrossed (Supplementary Table S3). Eggs were collected six times, with an interval of one to two days.
Crossing of tra2 ts2 G 2 : phenotypic males and females heterozygous for tra2 ts2 were inbred (Supplementary  Table S4). Additionally, four tra2 ts2 heterozygous XY males, not capable of coiling and storing their distiphallus, were group backcrossed. tra2 ts2 homozygous XY males were either backcrossed or crossed with heterozygous tra2 ts2 females (Supplementary Table S4). Nine males homozygous for tra2 ts2 with XX-karyotype, all not able to coil and store their distiphallus, were individually backcrossed to four females each. Eggs of the G 2 crosses were collected four to seven times over seven to 13 days (Supplementary Table S4).
Dissections: G 0 flies and single crossed G 2 flies were allowed to mate for 5-10 days (G 0 ) or 7-13 days (G 2 ) days. If still alive, they were then dissected to examine their internal reproductive organs.

Molecular analyses of G 0 mosaics.
To analyse the mosaic genotype of G 0 flies, DNA was extracted from single flies according to a standard protocol. The target region encompassing the ts1 and ts2 mutant sites (1213 bp) was amplified using the tra2-specific primers P1401 (TGC TTG GTG GTC CGC AAA TA) and P1500 (TGT GCA TAT ACT AAA GGC TCT CCC ), 50-100 ng DNA, and the Q5 High-fidelity DNA polymerase (New England Biolabs) according to the manufacturer's protocol in a Bio-Rad C1000 Touch thermal cycler [98 °C, 1 min; 35 cycles of (98 °C, 15 s; 56 °C, 30 s; 72 °C, 45 s); 72 °C, 2 min]. PCR fragments were purified using the Zymo Research DNA Clean & Concentrator -5 kit and subcloned into the pCR4-blunt TOPO vector (Invitrogen) for sequencing. Three to five clones were sequenced using primer mfs13 (TGT AAA ACG ACG GCC AGT ) (Macrogen Europe, Amsterdam) for each analysed fly. Verification of CRISPR-induced mutations from the sequencing results was performed using the Software Package Geneious Prime 50 by mapping the sequencing results to the tra2 reference sequence (Gene ID: 101,452,698).
Non-lethal genotyping of G 1 and G 2 flies. To identify the tra2 genotype of G 1 and G 2 flies, non-lethal genotyping was performed using an adapted version of the protocol established by Carvalho et al. 52 . A single leg of an anesthetized fly was cut at the proximal femur using scissors and homogenized in 50 µl buffer (10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl) for 15 s (6 m/s) using ceramic beads and a FastPrep-24 5G homogenizer (M.P. Biomedicals). 28.3 µl buffer mixed with 1.7 µl proteinase-K (2.5 U/mg) were added and incubated for 1 h at 37 °C. The reaction was stopped 4 min at 98 °C. The solution was cooled down on ice and directly Molecular karyotyping-Y chromosome specific PCR. Y-specific repetitive elements were amplified from genomic DNA extracted either from a single fly (G 0 ) or a single leg (G 2 ) using the published Y-specific oligonucleotides P1504_Y-spec1 (TAC GCT ACG AAT AAC GAA TTGG) and P1505_Y-spec2 (GCG TTT AAA TAT ACA AAT GTGTG) 53 . 10 µl PCR reactions contained either 50 ng DNA (single fly) or 3.75 µl single-leg DNA template solution, and the Y-specific primers and DreamTaq PCR components as described above. PCR cycling conditions (Bio-Rad C1000 Touch) were [95 °C, 3 min; 35 cycles of (95 °C, 30 s; 58 °C, 30 s; 72 °C, 1 min); 72 °C, 5 min]. Absence of a PCR product was interpreted as absence of the Y chromosome (XX-karyotype).
The same PCR conditions with primers P1532 (AGT GAA AAC GAT TTA AAT CAC ATG CAC) and P1500 for genomic DNA extracted from a single-leg, or P1401 and P1500 for DNA extracted from a single fly were used to amplify 328 bp or 1,213 bp fragments, respectively of tra2 as a positive control PCR to confirm sufficient quality of extracted genomic DNA.
Equipment and settings for image acquisition. For bright field image acquisition of flies (either dead or anesthetized with CO 2 and placed on a 4 °C cooler) was carried out using a fully automated Leica M205FC stereo microscope with a PLANAPO 1.0 × objective, a Leica DFC7000 T camera and the Leica LAS X 3.4.2.18368 software. To enhance screen and print display of the pictures the image processing software Fiji ImageJ Version 2.0.0 54 was used to apply moderate changes to image brightness and contrast. Changes were applied equally throughout the entire image and across all images.