Unless otherwise specified, all PCR amplifications were performed using Phusion DNA polymerase and Phusion-HF buffer (New England Biolabs GmbH, D-65926 Frankfurt am Main). Routine plasmid min-preps and PCR products were purified using NucleoSpin® Plasmid and NucleoSpin® Gel and PCR Clean-up kits (Macherey-Nagel GmbH & Co., 52355 Dueren, Germany), respectively. Plasmid vectors for microinjections were prepared using NucleoSpin® Plasmid Transfection-grade (Macherey-Nagel) or QIAGEN Plasmid Plus Midi Kit (QIAGEN GmbH, 40724 Hilden, Germany). Primers used are listed in Additional File 1: Supplementary Table 1.
Fly strain and husbandry
All fly experiments were performed in our well-equipped safety level one (S1) laboratory, which is certified for generating and using genetically modified insects. Wild type D. suzukii from Italy (kindly provided by Prof. Marc F. Schetelig) as well as generated transgenic lines were reared on standard Drosophila food supplemented with baker yeast and kept at 25℃ throughout this study. For germline transformation, flies were transferred to Drosophila egg laying cages and allowed to lay eggs on apple juice agar plates with some yeast on top to increase egg laying.
Nucleic acid isolation
Genomic DNA was isolated from a mix of adult males and females of D. suzukii (Italian strain) using NucleoSpin® DNA Insect (Macherey-Nagel) according to the manufacturer instructions. To generate a testes-specific cDNA library, testes of 100 males (3-4 days old) were dissected in ice cold 1X PBS and used for total RNA preparation using ZR Tissue & Insect RNA MicroPrep (Zymo Research Europe, 79110 Freiburg) according to manufacturer instructions.
Isolation of DsU6 and hsp70 genes
Based on synteny we identified D. suzukii the homologs of D. melanogaster genes Esyt2 and REPTOR bordering the U6 locus. Primer pair HM#137/138 was designed on the conserved parts of these genes and used to PCR amplify the sequence between them supposedly containing the Ds_U6 locus, (initial denaturation temperature 98℃ 3min followed by 35 cycles of 98℃ 30sec, 72℃ 2 min 30 sec). A 3.7 kbp fragment was obtained and sequenced.
To identify the D. suzukii heat shock protein 70 (Dshsp70) gene, we BLASTed D. melanogaster hsp70Aa in the D. suzukii genome data base (www.spottedwingflybase.org) and compared the amino acid sequence as well as the corresponding DNA sequence individually to their D. melanogaster counterparts using the geneious program version 10.2.6 (Auckland, 1010, New Zealand).
Isolation of Dsβ2t gene and its 5’UTR
To isolate the spermatogenesis specific beta-2-tubulin (β2t) gene of D. suzukii, we searched in the www.spottedwingflybase.org with the D. melanogaster Dm_β2t gene. A putative Ds_ β2t gene sharing high homology to Dm_β2t was PCR amplified from genomic DNA using primer pair HM#25/26 and the PCR program 98℃ for 3 min followed by 35 cycles of 98℃ 30 sec, 72℃ 1 min 40 sec, and 7 min final elongation at 72℃. The amplified fragment was purified, blunt cloned into pJet1.2 vector (Thermo Fisher Scientific, 64293 Darmstadt, Germany), and sequenced using standard primers pJet1.2_fwd and pJet1.2_rev.
Since the 5’UTR of β2t has some regulatory elements, whose position relative to the transcription start site and the upstream regulatory elements is highly conserved and important for correct tissue specific expression, it was imperative to isolate the 5’UTR and to identify the transcription start site. To do so, 1.7 µg of testes total RNA were used to generate a 5’ RACE-ready cDNA library using the SMARTerTM RACE cDNA amplification kit (Takara Bio Europe SAS, 78100 Saint-Germain-en-Laye, France) according to manufacturer instructions. The 5’UTR was recovered by RACE PCR using gene specific primer HM#33 and universal primer (UPM) provided with the kit using Advantage2 DNA polymerase (Takara) with the following program: 94℃ 2 min, (94℃ 30 sec, 72℃ 3 min) 5X, (94℃ 30 sec, 70℃ 30 sec, 72℃ 3 min) 5X, (94℃ 30 sec, 68℃ 30 sec, 72℃ 3 min) 30X. A single prominent band was recovered, purified, cloned into pCRII (Thermo Fisher Scientific) to generate pCRII_Dsb2t_5’UTR (HMMA24), and sequenced using a standard M13 primer.
Testes whole mount in situ hybridization
To generate DIG-labelled sense and antisense RNA probes of Ds_β2t, we prepared DNA templates for in vitro transcription by PCR amplification of the 5’RACE-fragment including the Sp6 or T7 promoters from pCRII_Ds β2t_5’UTR (HMMA24). Primer pairs HM#33/128and HM#41/127 were used respectively with the following PCR conditions: initial denaturation at 98℃ 3 min, followed by 35 cycles of 98℃ 30 sec, 72℃ 50 sec with a final elongation step of 7 min. RNA probes were synthesized using DIG-labelling kit (Thermo Fisher Scientific) according to manufacturer instructions using 200ng of DNA as template in a total reaction mix of 10µl. The reaction was allowed to proceed for 2h at 37℃ followed by Turbo DNaseI treatment (Thermo Fisher Scientific) for 15 min to remove template DNA. 2µl of 0.2M EDTA was used to inactivate the reaction. Sense and antisense probes were precipitate and resuspended in 100µl RNA resuspension buffer (5:3:2 H2O: 20X SSC: formaldehyde) and stored at -80℃.
Testes of 3-5 days old males were dissected in ice cold 1X Phosphate buffered saline (PBS) and fixed in PBF-tween (4% formaldehyde and 0.1% tween 20 in 1X PBS) for 20 min at room temperature. In situ hybridization was performed according to an established protocol (56) with inclusion of dehydration steps according to Zimmerman et al. (57).
Plasmid construction
To generate plasmid HMMA006, 300 bp upstream of Ds_sryα plus 50bp 5’UTR sequence were PCR amplified using primer pair HM#23/24 introducing AgeI/NheI cut sites respectively and cloned into AgeI/NheI cut site of KNE007 (58) upstream of tTA CDS replacing the Dm_β2t promoter. Description of the Ds_sryα gene and its cloning will be described elsewhere (Ahmed et al.)
To generate pSLaf_T7-BbsI-BbsI-ChiRNA_af (HMMA034) for in vitro transcription of gRNAs, annealed oligos HM#55/56 generating T7 promoter and 2X BbsI restriction sites were cloned into BbsI/HindIII digested plasmid pU6-chiRNA (Addgene: #45946) giving rise to HMMA033. Next, the HindIII/SacI T7-BbsI-BbsI-chiRNA fragment from HMMA033 was cloned into pSLaf1180af (59) HindIII/SacI cut sites.
To generate plasmids pDsU6a-BbsI-BbsI-chiRNA-DSE (HMMA091), pDsU6b-BbsI-BbsI-ChiRNA DSE (HMMA092), and pDsU6c-BbsI-BbsI-chiRNA-DSE (HMMA093) for transient expression of gRNAs, primer pairs HM#358/159, HM#104/158, and HM#360/160 were used to amplify the promoters of snRNA genes U6a, U6b, and U6c, respectively, with PCR condition 98℃ 3 min followed by 5 cycles of 98℃ 30 sec, 66℃ 40 sec, and 72℃ 1 min then 30 cycles of 98℃ 30 sec, 72℃ 1 min 40 sec with a final elongation 72℃ for 7 min. The promoters were then cloned into HMMA034 by megaprimer PCR cloning (60) using 30ng of plasmid HMMA034 and 200ng of the promoter as megaprimer in a 25µl reaction with PCR (98℃ 3 min, [98℃ 30 sec, 72℃ 2min 30 sec] 30X, 72℃ 7min) generating plasmids HMMA088, HMMA089, and HMMA090. Finally, 250 bp of the sequence downstream of the U6c termination sequence was PCR amplified from genomic DNA using primer pair HM#186/187 with PCR (98℃ 3 min, [98℃ 30 sec, 68℃ 30 sec, 72℃ 20 sec] 35X with a final elongation of 7 min at 72℃). The amplified fragment was then cloned into HMMA088, HMMA089, and HMMA090 by megaprimer cloning as described above with annealing temperature at 68℃.
For Cas9 recombinant protein expression, the plasmid pET-T7-3XFlag-nls-Cas9-nls-6XHisTag (HMMA101) was generated. The sumo part of the pET-SUMO expression vector was removed using XhoI/NdeI and the annealed oligos HM#152/153 were cloned introducing 2X BsaI sites giving rise to HMMA080. The 4.3Kb BbsI/XbaI 3XFlag-nls-Cas9-nls fragment was excised from HMMA066 and cloned into BsaI linearized HMMA080 to give rise to HMMA099. Finally, annealed oligos HM#180/181 introducing a 6XHisTag were cloned into FseI/BasI digested plasmid HMMA099. Plasmid HMMA066 was generated by cloning ClaI/HpaI fragment 3XFlag-nls-Cas9-nls from HMMA039 into ClaI/HpaI cut #1215 (20) giving rise to HMMA065 followed by cloning of annealed self-complementary oligo HM#102 into the ClaI site of HMMA065 to introduce 2X BbsI restriction sites. Cas9 protein was expressed and purified according to Paix et al. (61), and frozen at -20℃ until needed.
The plasmid pSLaf_Dshsp70P-Cas9-SV40_af (HMMA056) to express Cas9 transiently was generated by cloning of the 4.2Kb ClaI/XbaI fragment containing insect codon optimized Cas9 CDS with N and C terminal nuclear localization signals from plasmid #46294 (Addgene) into ClaI/XbaI digested pCS2-Sp6-Cas9-SV40 (Addgene: #47322) replacing the mammalian codon optimized Cas9 CDS giving rise to HMMA039. The Ds_hsp70 promoter was PCR amplified from genomic DNA using primer pair HM#73/75 with PCR using the following condition: 98℃ 3 min [(98C℃ 30 sec, 66℃ 40 sec, 72℃ 1min) 5X, (98℃ 30 sec, 72℃ 1 min 40 sec) 35X with a final elongation step of 7 min at 72℃. The fragment was purified and cloned into EcoRI/ClaI cut #1215 (20) to give rise to HMMA052. Finally, Cas9-SV40 was excised from HMMA039 by ClaI/HpaI and cloned into ClaI/HpaI cut HMMA052 generating HMMA056.
To generate donor plasmid HMMA134, a 3.2Kb fragment containing PUb-nls-EGFP-SV40 was excised from #1254 (20) using SacI/AflII and cloned into SacI/AflII cut pSLaf1108af (59) giving rise to plasmid HMMA094. DsRed CDS was PCR amplified from plasmid KNE007 (58) using primer pair (HM#37/167) with PCR (98℃ 3 min followed by 35 cycles of 98℃ 30 sec, 72℃ 1 min and a final elongation of 7 min at 72℃). The fragment was phosphorylated and ligated into blunted AflII cut HMMA095 generating HMMA096. To change the target PAM sequence in front of EGFP from TGG to TGA in the repair template (Figure 1B), PCR mutagenesis using primer pair HM#221/222 was performed (98℃ 3 min followed by 30 cycles of 98℃ 30 sec, 72℃ 4 min and final elongation of 7 min at 72℃) to give rise to HMMA097, which results in changing the second amino acid of the EGFP from valine to methionine. Finally, the 247 bp Ds_β2t regulatory sequence spanning -51 to +196 was PCR amplified using primer pair HM#285/252 with PCR conditions 98℃ 3 min [(98℃ 30 sec, 60℃ 30 sec, 72℃ 20 sec) 5X, (98℃ 30 sec, 72℃ 1 min) 30X with a final elongation step of 7 min at 72℃. The promoter was then cloned upstream of DsRed in HMMA097 by megaprimer PCR cloning as described previously with annealing at 61℃.
Guide RNAs design, cloning, and validation
Guide RNAs were identified using the online target finder tool built by Wisconsin University (http://targetfinder.flycrispr.neuro.brown.edu/). Identified potential targets were checked against D. suzukii database to exclude those with off-target sites. For each potential target, two oligos, a forward and reverse, were designed and the respective overhangs were added. Oligos were ordered as normal primers without phosphorylation. The two oligos for each target were annealed at a concentration of 10 µM in a total volume of 100 µl in a heat block. The gRNAs were validated using a T7EndoI assay (62,63). Each gRNA plasmid was mixed with Cas9 plasmid HMMA056 at a concentration of 400/500 ng/µl, respectively, and injected into 50 pre-blastoderm embryos. 10 – 15 hatching larvae were collected in 1.5 ml Eppendorf tubes and crushed by using a pipette tip against the tube wall. 200µl of squishing buffer (19) was added and mixed well. The tubes were then incubated at 55℃ for 1 h with occasional vortexing. Tubes were then centrifuged, and 5µl of the supernatant was used as a template in 50 µl PCR reactions using primers HM#192/69. PCR products were gel purified, quantified, and 400ng were mixed in 1X NEB 2.1 buffer in a total volume of 19 µl. DNA was denatured, rehybridized, 0.75 µl of T7 EndoI (NEB) were added, and incubated at 37℃ for 20 minutes. The reactions were stopped using 2 µl of 0.25M EDTA and run in a 1.5% agarose gel. Only one guide showed obvious digest by T7 EndoI. Wild type un-injected larvae were used as control. To generate the plasmids expressing the functional guide RNA against the identified target upstream of DsRed (Figure 1B), annealed oligos HM#161/162 and HM#169/162 were cloned by golden gate (64,65) into gRNA vectors HMMA091, HMMA092, and HMMA093 to generate pU6a_Red1chic HMMA102, pU6b_Red1chi HMMA103, and pU6c_Red1chi HMMA104, respectively.
In vitro transcription of the gRNA
The functional gRNA was cloned by ligation of annealed oligos HM#162/215 into BbsI cut plasmid HMMA035, which was then used to generate the template for in vitro transcription by PCR using primer pair HM#84/128. In vitro transcription of gRNA was performed using MEGAscript® (Ambion) according to the manufacturer protocol. The reaction was allowed to proceed for 2h at 37℃ followed by DNA template removal using 1 µl DNase I for 30 minutes. gRNA was purified using RNA clean and concentrator (Zymo Research) and the concentration was determined by nano-drop (Thermo Fisher Scientific) and stored at -80℃.
Germline transformation
All embryonic injections were performed using transfection grade plasmid preparations without further precipitation steps. To generate the embryonic driver line 06_F5M2 by random piggyBac integration, the transformation vector HMMA006 and the helper plasmid MK006 (58) were mixed at a final concentration of 400 and 200 ng/µl respectively. To validate that the transgene represents a single integration even, we performed inversePCR as described (58) using XhoI and EcoRI restriction enzymes. For both the 5 and 3’ junctions, we each obtained only a single fragment, whose sequences confirmed a single integration site in the second intron of a gene referred to as Suppressor of Under Replication (Additional File 2: piggyBac insertion in D. suzukii line 06_F5M2).
For the transgene editing experiments using CRISPR/Cas9, DNA was mixed at a concentration of 400, 150, and 350 ng/µl for Cas9 (HMMA056), gRNA (HMMA102, HMMA103, or HMMA104), and donor plasmid HMMA097, respectively. Higher concentration was used at 400, 250, and 400 ng/µl, respectively. All DNA injection mixes were prepared in 1X injection buffer (5mM KCl, 0.1 mM NaH2PO4, pH 6.8). For RNP injection, recombinant Cas9 endonuclease, gRNA, and donor plasmid HMMA097 were mixed together at a final concentration of 300 ng/µl, 150 ng/µl, and 400 ng/µl respectively, incubated at 37℃ for 10 minutes for the RNP-complex formation, and injected into 90 pre-blastoderm embryos.
Injection needles were prepared as previously described (58) .To inject in D.suzukii embryos, the eggs have to be squeezed out of the apple agar plates individually using home-made closed-tip glass pipettes. Embryos were then de-chorionated for 3 minutes using generic Clorox (DanKlorix, CP GABA GmbH, Hamburg, Germany) containing 2.5% sodium hypochlorite at final concentration of 1.25% sodium hypochlorite and washed in washing buffer (100mM NaCl, 0.02% Triton X-100) followed by thorough wash with desalted water. Embryos were then aligned on apple agar blocks and transferred to double sticky tape on a coverslip and covered by Voltalef 10S oil (VWR International, Darmstadt, Germany). Injections were performed using a Femtojet (Eppendorf, Hamburg, Germany) and a manual micromanipulator. Excessive oil was drained and the injected embryos were incubated on apple agar plates at the room temperature until hatching. Larvae were manually transferred to fly food vials. Each emerging G0 fly was out-crossed to 3-4 wild type individuals of the opposite sex.
Microscopy
Screening for transgenic flies and fluorescence imaging were performed using a Leica M205 FA fluorescence stereomicroscope equipped with camera Q imaging Micropublisher 5.0 RTV (Leica Mikrosysteme Vertrieb Gmb, Wetzlar, 35578 Germany). Transgenic flies were screened using filter sets RFP (excitation: ET546/10x, emission: ET605/70m) or GFP-LP (excitation: ET480/40, emission: ET510 LP), respectively, and imaged using cold light (Figure 1C) or filter sets: RFP (Figures 1D; 3F-H), EYFP (excitation: ET500/20, emission: ET535/30) for Figure 1E, or GFP-LP (Figure 3C-3E).
Epifluorescence microscopy was performed using a Zeiss Imager.Z2 equipped with two cameras, Axiocam 506 mono and Axiocam 305 colour (Zeiss, 73447 Oberkochen, Germany). The testes or the spermathecae were dissected in ice-cold PBS, fixed for 10 minutes in 4% formaldehyde prepared in 0.1% PBS-tween 20, permeabilized for 10 minutes using 1% Triton X-100 in PBS, and nuclei were stained for 10 minutes using DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) at a concentration of 1 µg/ml. Samples were mounted in 70% glycerol and the spermathecae were broken open using dissection needles. The tissues were imaged under bright field and to observe cell nuclei and expression of DsRed, images were taken with filters for DAPI (excitation: 335-383, emission: 420-470) or DsRed (excitation: 533-558, emission: 570-640), and composed in ZEN Blue (Zeiss).