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[(stages\))-332.8(were)-331.2(dissected,)-331.4(frozen)-328.6(in)-330(liquid)-334.3(nitrogen,)-326.3(placed)]TJ
0 -1.1923 TD
[(in)-460.1(RNAlater)-455.5(\(Thermo)-453.7(Fisher)-455.7(Scientic\),)-452.4(and)-455.3(stored)-458.9(at)]TJ
/F12 1 Tf
T*
()Tj
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(80)Tj
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()Tj
/F7 1 Tf
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[(C.)-590.6(Total)-592.2(RNA)-591.5(was)-596.2(extracted)-593(with)-588.4(the)-593(RNeasy)]TJ
-2.1678 -1.1869 TD
[(Mini)-340.1(Kit)-340.5(\(Qiagen\),)-337(followed)-336.7(by)-338.6(removal)-336.7(of)-337.6(gDNA)-343(with)]TJ
0 -1.1923 TD
[(DNase)-379.6(I)-371.6(\(Thermo)-372.4(Fisher)-374.4(Scientic\).)-371.1(Puried)-374(RNA)-374.7(was)]TJ
T*
[(diluted)-456.3(to)-449.2(20)-165.2(ng/)]TJ
/F13 1 Tf
6.9263 0 TD
(l)Tj
/F7 1 Tf
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[(L)-453.7(and)-455.3(used)-449.6(to)-454.7(produce)-452.9(cDNA)-450.2(pools)]TJ
-7.4736 -1.1869 TD
[(with)-507.1(the)-500.8(RevertAid)-504.1(First)-503.8(Strand)-497.8(cDNA)-504.4(Synthesis)-504.7(Kit)]TJ
0 -1.1923 TD
.016 Tc
[(\(Thermo)-540.8(F)4.4(is)5.4(her)-539.8(S)4.4(cienti)4.3(c)]TJ
11.2023 0 TD
[(\).)-536.1(RT-PCR)-537.4(was)-536.6(c)-3.1(ond)6.6(ucted)]TJ
-11.2023 -1.1923 TD
0 Tc
[(using)-397.5(the)-392.5(Q5)-391.6(High-Fideli)-8.5(ty)-389.6(2)]TJ
/F10 1 Tf
12.3188 0 TD
()Tj
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[(Master)-397.9(Mix)-393.4(\(NEB\).)-391.9(All)]TJ
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0 -1.1923 TD
[(ment)-7.1(ary)-302.5(Table)-301.6(S1.)]TJ
/F9 1 Tf
/Cs8 cs 1 scn
0 -2.3684 TD
[(Construct)-305.4(design)]TJ
/F7 1 Tf
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[(Adult)-267.2(ovary)-267.2(total)-269.4(RNA)-271.8(was)-265.6(used)-270.8(to)-265(amplify)-269.6(the)-267.8(5)]TJ
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()Tj
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[(and)-271(3)]TJ
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()Tj
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-22.6106 -1.1923 TD
[(UTR)-392(of)]TJ
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(Pxmeiw68)Tj
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(and)Tj
/F11 1 Tf
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(PxnanosP)Tj
/F7 1 Tf
4.4495 0 TD
[(using)-386.6(the)-392.5(SMARTer)]TJ
-14.4704 -1.1869 TD
[(RACE)-392.6(5)]TJ
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()Tj
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()Tj
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[(Kit)-389.2(\(Takara\).)-387.8(Regulatory)-384.7(sequences)-391.5(5)]TJ
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()Tj
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(and)Tj
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(3)Tj
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[(were)-520.9(then)-526.5(cloned)-522.5(from)-524.3(fourth)-522.9(instar)-526.1(larval)-523.8(gDNA)]TJ
-1.279 -1.1923 TD
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[(\(NucleoSpin)-425.7(Tissue)-423.1(Column;)-420.3(Macherey-Nagel\).)]TJ
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(Piggy-)Tj
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(Bac)Tj
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[(constructs)-536.8(AGG1906)-528.5(\()]TJ
/F11 1 Tf
9.6144 0 TD
(Pxmeiw68-Cas9)Tj
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[(;)-537.4(GenBank)]TJ
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[(P)12.4(x)0(n)20.3(a)12.2(no)19.1(s)11(P)0(-)]TJ
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[(Ca)17.1(s)11(9)]TJ
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[(;)-304.3(GenBank)-319.2(accession)-317.8(numb)-6.1(er)-309.3(OK145567)-8(\))-311.9(were)-315(de-)]TJ
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[(ve)-6.8(loped)-465.5(as)-468.7(for)-465.3(AGG1536)-468.9(\()]TJ
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(Pxvasa-Cas9)Tj
/F7 1 Tf
5.2841 0 TD
[(\))-469.1(previously,)]TJ
7.8454 0 0 6.9729 544.3085 441.3542 Tm
(26)Tj
10.4607 0 0 10.4607 313.0582 424.2329 Tm
[(ex)-6.8(cept)-514.5(for)-519.5(repla)-8.1(cing)-515.7(the)]TJ
/F11 1 Tf
10.8338 0 TD
[(Pxva)-8.1(sa)]TJ
/F7 1 Tf
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[(promote)-7.3(r,)]TJ
/F11 1 Tf
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(Pxvasa)Tj
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(3)Tj
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/F7 1 Tf
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[(UTR,)-391.3(an)-6.8(d)-383.4(SV40)-391(termina)-8.3(tor)-387.2(with)-393.2(corresp)-8.7(onding)-389.4(regula-)]TJ
0 -1.1869 TD
[(t)-6.2(ory)-428.4(frag)-7.5(ments)-435(from)]TJ
/F11 1 Tf
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(Pxmeiw68)Tj
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(and)Tj
/F11 1 Tf
1.8752 0 TD
[(Pxnan)-7.9(osP)]TJ
/F7 1 Tf
4.4983 0 TD
[(as)-436.2(well)]TJ
-19.8629 -1.1923 TD
[(as)-235.7(the)-235.3(uoropho)-6.2(re)-233.5(marker)-237.3(3)]TJ
/F10 1 Tf
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()Tj
/F7 1 Tf
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[(UTR)-234.9(with)-236.1(P10.)-235.8(All)-232.1(plasmi)-7.7(ds)]TJ
-11.495 -1.1869 TD
[(wer)-8.6(e)-431(assemb)-8.7(led)-435.8(with)-436.6(NEBuilder)-440.1(HiFi)-433.4(DNA)-436.6(Assemb)-8.6(ly)]TJ
0 -1.1923 TD
[(Cl)-6.8(oning)-298.7(Kit)-302.5(\(NEB;)-309.1(see)-303.5(Fig.)-297.6(1A\).)]TJ
1.0514 -1.1923 TD
[(For)-457.7(constructing)-459.1(sgRNA)-459.9(expressing)-458.7(lines)-463.5(\()48.2(homing)]TJ
-1.0514 -1.1869 TD
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[(e)-5.8(l)1.6(ements)48.2(\),)-311.2(two)-315.9(p)3.8(igmentation)-310.5(g)-1.6(enes)]TJ
/F11 1 Tf
14.1398 0 TD
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[(Pxye)-6.4(llow)]TJ
/F7 1 Tf
3.7558 0 TD
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(and)Tj
/F11 1 Tf
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(Pxkmo)Tj
/F7 1 Tf
7.8454 0 0 6.9729 544.6487 341.7448 Tm
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(27)Tj
10.4607 0 0 10.4607 313.0582 324.6235 Tm
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[(were)-369.2(selected)-363.6(as)-365.7(targets)-366.4(for)-362.3(CRISPR-b)8.2(ased)-367.1(site-s)9.2(pecic)]TJ
0 -1.1923 TD
[(insertion)-442.9(\()48.2(knock)10.6(-in)48.7(\))-445(of)-451.4(sgRN)9(A)-452.6(casset)7.4(tes)-447.8(due)-449.9(to)-449.2(the)]TJ
0 -1.1869 TD
[(ease)-420.2(of)-413.5(visual)9.4(ly)-416.7(scoring)-408.7(null)-410.7(mutation)10.3(s)-417.1(i)0(n)-416.7(these)-411.3(genes.)]TJ
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T*
[(immediate)7.9(ly)-481.8(anking)-475.8(each)-484(sgRNA)-476.1(cleavage)-478.2(site)-481.2(were)]TJ
0 -1.1869 TD
[(cloned)-511.7(from)-508.1(gDNA)-511(as)-512.1(homology)-506.7(arms)-509.1(for)-508.7(mediating)]TJ
0 -1.1923 TD
[(HDR)-293.5(repair.)-292.2(Six)-287.5(RNA)-293.4(polymeras)11.8(e)-295.5(III)]TJ
/F11 1 Tf
15.4947 0 TD
(PxU6)Tj
/F7 1 Tf
2.5689 0 TD
[(promot)11.8(ers)]TJ
7.8454 0 0 6.9729 544.3652 254.5511 Tm
(32)Tj
10.4607 0 0 10.4607 313.0582 237.4299 Tm
[(were)-347.5(divided)-340.3(into)-345.7(two)-343.7(sets)-350.1(of)-343(three)-344.1(and)-346.9(used)-346.7(to)-346.3(express)]TJ
0 -1.1869 TD
[(relevant)-454.7(sgRN)9(As)-457.8(for)-454.5(each)-451.5(gene)-452.7(\(see)-452.9(Fig.)-454.8(1B\).)-451.3(Within)]TJ
0 -1.1923 TD
[(each)-603.2(c)13.4(o)17.7(n)17.7(s)11(t)15.4(r)18.7(u)12.2(c)13.4(t)15.4(,)-584.6(a)13.4(l)15.4(t)15.4(h)12.2(o)17.7(u)17.7(g)12.2(h)-583.9(m)11.4(u)]TJ
12.0586 0 TD
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[(ltiple)-603.7(U6)-604.8(promoters)-600.6(w)-2(ere)]TJ
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[(used,)-284.8(o)-2.3(nly)-284.1(a)-282.9(single)-282.9(sgRNA)-282.9(w)-1.1(a)4.3(s)-285.3(expr)4.2(essed)-284.1(\()4.2(all)-286.3(U)4.4(6)-284.1(p)-2.3(r)4.2(o)-2.3(mot-)]TJ
0 -1.1869 TD
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[(er)15.8(s)-216.6(w)0(i)18.1(t)0(h)22.3(i)0(n)-210.8(t)0(h)22.3(e)-225(s)11(a)13.4(me)-213.6(c)13.4(o)0(n)19.1(s)11(tr)17.8(u)12.2(c)0(t)-209.6(e)0(x)20.2(p)0(r)20.1(e)0(s)19.1(s)11(ed)-212.8(th)22.3(e)-219.6(s)0(a)19.1(m)11.4(e)-225(s)11(g)12.2(R)10.2(NA)21.5(\).)]TJ
0 -1.1923 TD
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[(sgRNAs)-528.1(were)-525.7(d)-1.2(esigned)-526.9(w)0(ith)-526.9(C)2.2(HOPCHOP)-529.1(\(https://)]TJ
T*
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[(ch)14.8(o)12.2(p)12.2(c)0(h)20.2(o)12.2(p.)18.4(cb)20.3(u)12.2(.)0(u)18.4(i)0(b)22.3(.)11.5(no)19.1(/\))17.8(.)-340.7(s)11(g)12.2(R)10.2(NA)-330.8(ex)20.3(p)12.2(r)0(e)15.8(s)11(s)11(i)0(o)22.3(n)-340(ca)16(s)11(s)11(e)0(t)18(t)0(e)18(s)-335.8(we)16.1(r)13.2(e)]TJ
0 -1.1869 TD
[(sy)17.9(n)12.2(t)0(h)16.8(e)13.4(si)15.6(z)13.4(e)0(d)-402.5(a)13.4(nd)-398.3(cl)18(o)12.2(n)0(e)20.3(d)-410.5(in)22.3(to)-400.5(th)22.3(e)-409.3(h)12.2(om)18.2(o)12.2(l)0(o)16.8(g)12.2(y)-410.5(a)13.4(rm)-398.1(de)20.2(st)15.7(in)22.3(a-)]TJ
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[(t)10(i)30(o)10(n)-480.4(plasmid,)-509.6(alongside)-507.7(a)-507.7(Z).7(sgreen-expressing)-508.9(m)10(a)-8.5(rker)]TJ
T*
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[(\(Fig.)-257.1(1)3.7(B;)-258.6(Genewiz;)-258.6(GenBank)-261.8(accession)-256.4(numbers)-257.6(AGG1619:)]TJ
0 -1.1869 TD
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[(OK1)5.3(455)5.3(68,)-694.5(AGG1)5.3(962)5.3(:OK1)5.3(455)5.3(70,)-699.9(an)5.3(d)-699.3(AGG196)5.3(3:)]TJ
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[(OK1)5.7(455)5.7(69\).)]TJ
1.0514 -1.1923 TD
0 Tc
[(Assembled)-645(constructs)-650.6(were)-651(puried)-648.2(with)-648(Nucleo-)]TJ
-1.0514 -1.1869 TD
[(Bond)-381.2(Xtra)-378(Midi)-378(Prep)-373.8(Kit)-378.4(EF)-378.6(\()-84.3(Macherey-Nagel\))-376(before)]TJ
0 -1.1923 TD
(injection.)Tj
/F5 1 Tf
9.9626 0 0 9.9626 62.022 754.3557 Tm
(2)Tj
44.8301 0 TD
[(XU)-306.7(ET)-299.4(AL.)]TJ
ET
endstream
endobj
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<>stream
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BT
/F9 1 Tf
10.4607 0 0 10.4607 59.7543 698.2297 Tm
/Cs8 cs 1 scn
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[(Development)-298.3(of)-300.7(transgenic)-300(lines)]TJ
/F7 1 Tf
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(A)Tj
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(Pxvasa-Cas9)Tj
/F7 1 Tf
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[(line)-317.4(\(1536A\))-317.6(was)-319.8(built)-319.4(previously.)]TJ
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(26)Tj
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(To)Tj
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[(mitigate)-429.7(issues)-429.8(associated)-432.4(with)-425.8()45.8(positional)-426(effects)46.9()-431.2(o)0(f)]TJ
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/F11 1 Tf
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[(Px)20.4(va)20.3(sa)17.9(-)13.2(C)0(a)17(s)11(9)]TJ
/F7 1 Tf
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[(li)20(n)12.2(e)0(s)]TJ
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[(were)-325.8(genera)-8.7(ted)-322(here,)-326.3(initially)-324.7(using)-327(the)-322(same)-324.6(method)-324(as)]TJ
T*
[(previo)-7.9(usly)-257.9(describe)-9.3(d.)-258(Subsequ)-8.6(ently,)-261.1(to)-259.6(improve)-262(transf)-8.1(or-)]TJ
0 -1.1869 TD
[(matio)-7.9(n)-350.9(efcien)-11.7(cy,)]TJ
/F11 1 Tf
7.7175 0 TD
[(piggyB)-8.7(ac)]TJ
/F7 1 Tf
4.1514 0 TD
[(helper)-358.9(plasmi)-7.7(d)-350.9(and)-357.7(codon-)]TJ
-11.869 -1.1923 TD
[(optimiz)-7.3(ed)-472.9(mRNA)-471.7(were)-472.1(repla)-8.1(ced)-470.3(with)-474.5(a)-468.9(comme)-8.5(rcially)]TJ
T*
[(synth)-7.2(esized)]TJ
/F11 1 Tf
4.9156 0 TD
(piggyBac)Tj
/F7 1 Tf
4.0214 0 TD
[(mRNA)-233.3(\(TriL)-9.1(ink)-231.1(Biotechnologies\))-12.2(.)]TJ
-8.9369 -1.1869 TD
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[(Constructs)-615.1(\(AGG1906)-614(o)-1.5(r)-618.4(AGG2093\))-618.4(were)-618.2(i)1.7(njected)-619.4(at)]TJ
0 -1.1923 TD
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[(500)-162.6(ng)5.4(/)]TJ
/F13 1 Tf
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(l)Tj
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[(L)-242.4(a)2.5(lo)6.7(ngs)5.5(id)6.7(e)-246.9(6)1.3(0)6.7(0)-166.8(n)6.7(g)1.3(/)]TJ
/F13 1 Tf
7.5875 0 TD
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(l)Tj
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(L)Tj
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[(pigg)4.8(yBac)]TJ
/F7 1 Tf
3.9238 0 TD
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[(mR)-4.8(NA)-288.8(to)-290(gene)-20.4(r-)]TJ
-16.2534 -1.1923 TD
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[(at)18(e)]TJ
/F11 1 Tf
1.6259 0 TD
[(P)12.4(x)0(v)21.4(a)0(s)17.9(a)12.2(-C)18.1(a)12.2(s)11(9)]TJ
/F7 1 Tf
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(,)Tj
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[(P)12.4(x)0(m)21.5(e)0(i)18(w)10.2(68)19.1(-)13.2(C)10.2(as)17.9(9)]TJ
/F7 1 Tf
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[(,a)465.4(n)485.7(d)]TJ
/F11 1 Tf
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[(P)-12.2(x)2.8(nano)-12.2(s)5.7(P).2(-)-12.2(C)5.9(as)-12.2(9)]TJ
/F7 1 Tf
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[(li)20(ne)20.3(s.)]TJ
1.0514 -1.1923 TD
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[(For)-201.8(c)-1(onst)-4.5(ruct)-4.5(ing)-197.4(homing)-202.8(e)-1(lement)-4.5(s,)-203.5(sgRNAs)-204(were)]TJ
/F11 1 Tf
18.9741 0 TD
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[(in)-200(vi)18(tr)21.1(o)]TJ
/F7 1 Tf
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[(tr)17.8(an)20.3(s)11(c)0(r)15.8(i)0(b)22.3(e)0(d)-288.7(\()0(1)20.1(5)12.2(0)-155.8(n)0(g)19.1(/)]TJ
/F13 1 Tf
7.8639 0 TD
(l)Tj
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[(L\))-13(,)-302.6(c)-13(o)7.3(m)-1.6(p)-13(l)3.8(ex)-13(e)7.3(d)-315.1(w)-13(i)5.1(th)-315.1(30)-13(0)-162(ng/)]TJ
/F13 1 Tf
11.0289 0 TD
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(l)Tj
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[(LC)312.1(a)315.3(s)301.9(9)]TJ
-19.9767 -1.1869 TD
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[(pr)20.1(ot)16.8(e)13.4(i)0(n)]TJ
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(27)Tj
10.4607 0 0 10.4607 99.2126 498.9542 Tm
-.0129 Tc
[(a)-12.9(n)7.4(d)-271.6(i)-12.9(nj)-12.9(e)10.6(c)-12.9(t)5.1(e)-12.9(d)-263.6(a)-12.9(l)5.1(ongs)-12.9(ide)-275.9(t)-12.9(h)9.4(e)-270.4(r)-12.9(e)2.9(l)-12.9(e)10.6(v)-12.9(a)7.4(n)-12.9(t)-267(sgRN)-12.9(A)-262.4(p)-12.9(las-)]TJ
-3.7721 -1.1923 TD
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[(mi)16(d)-443(\()0(8)20.1(0)12.2(0)-155.8(n)0(g)19.1(/)]TJ
/F13 1 Tf
5.1974 0 TD
(l)Tj
/F7 1 Tf
.542 0 TD
[(L)12.4(\))0(.)-441.3(I)13.2(nj)16.8(e)13.4(c)0(t)18(i)0(o)22.3(n)0(,)-436.9(t)0(r)17.8(a)13.4(ns)17.9(fo)20.1(r)13.2(m)0(a)19.4(n)0(t)16.8(,)-443.7(an)20.3(d)-448.4(i)0(n)22.3(s)11(er)15.8(ti)20(o)12.2(n)12.2(-)]TJ
-5.7394 -1.1923 TD
[(si)15.7(te)-280(id)22.3(en)20.3(ti)20(c)18.1(a)13.4(t)0(i)20(o)0(n)-279(w)0(e)16.1(r)13.2(e)-290.1(a)0(s)-284.4(p)12.2(r)13.2(e)0(v)20.3(i)0(o)16.8(u)12.2(s)11(l)0(y)-281.2(d)12.2(es)19.1(cr)15.8(i)15.4(b)0(e)14.8(d)12.2(.)]TJ
7.8454 0 0 6.9729 256.9889 478.6581 Tm
-.0158 Tc
[(25)-15.8(,)11.6(2)-15.8(7)]TJ
10.4607 0 0 10.4607 70.7527 461.5936 Tm
0 Tc
[(For)-403.5(resolving)-408.1(the)-403.3(5)]TJ
/F10 1 Tf
8.059 0 TD
()Tj
/F7 1 Tf
.6558 0 TD
[(junction)-406.1(between)-404.9(the)-408.7(integrated)]TJ
-9.7661 -1.1923 TD
[(homology)-479.6(arm)-482.2(and)-482.4(the)-484.6(genomic)-478.6(anking)-481.2(sequence)-483.8(in)]TJ
T*
[(1619P15,)-452.4(the)-452.1(Cas9)-457.1(Sequencing)-447.5(Kit)-454.3(\(Oxford)-452(Nanopore)]TJ
0 -1.1869 TD
[(Technologies\))-486.7(was)-487.8(used)-482.2(because)-485.2(PCR)-484.4(could)-487.2(not)-480.4(con-)]TJ
0 -1.1923 TD
[(rm)-702.4(the)-706.8(junction.)-703.5(In)-700.7(brief,)-701.3(high)-706.6(molecular)-703.4(weight)]TJ
T*
[(\(HMW\))-209.2(genomic)-207.6(gDNA)-207.5(was)-205.9(extracted)-208.2(from)-204.6(homozygous)]TJ
0 -1.1869 TD
[(1619P15)-344.7(pupae)-342.9(using)-343.3(the)-349.1(Monarch)-344.3(HMW)-344.1(DNA)-344.4(extrac-)]TJ
0 -1.1923 TD
[(tion)-546.2(kit)-547.6(for)-541.2(tissue)-550(\(NEB\).)-543.7(The)-547.2(Cas9)-543.8(Sequencing)-545(Kit)]TJ
T*
[(\(Oxford)-424.9(Nanopore)-417.7(Technology\))-422.2(was)-422.7(used)-422.5(to)-422.2(prepare)-423.6(a)]TJ
0 -1.1869 TD
[(library)-495.4(targeting)-494.7(the)-495.4(insertion.)-490.9(Targets)-494(GAACTCGGT)]TJ
0 -1.1923 TD
(GATGACGTTCT)Tj
ET
136.97 334.545 21.997 .45355 re
f
BT
10.4607 0 0 10.4607 136.97 337.0393 Tm
[(CGG)-710.8(and)-710(GCTGAAGGGCG)5.1(AGAC)]TJ
-7.3815 -1.1923 TD
(CCACA)Tj
ET
95.698 322.072 22.507 .51022 re
f
BT
10.4607 0 0 10.4607 95.6976 324.5668 Tm
[(AGG)-398.6(were)-401.7(designed)-401.7(to)-400.5(target)-399.1(the)-403.3(DsRed)-400.3(open)]TJ
-3.436 -1.1869 TD
[(reading)-514.7(frame)-515.1(using)-511.3(CHOPCHOP)-512.6(and)-514.9(synthesized)-513.5(as)]TJ
0 -1.1923 TD
[(Alt-R)-430.8(CRISPR-Cas9)-423.1(crRNAs)-429.1(\(IDT\).)-426.9(These)-430.6(along)-422.2(with)]TJ
T*
[(the)-479.2(Alt-R)-479.6(CRISPR-Cas9)-477.3(tracrRNA)-473.9(and)-477(Alt-R)-479.6(SpCas9)]TJ
0 -1.1869 TD
[(Nuclease)-614.9(V3)-613.8(\(IDT\))-611.8(were)-618.5(used)-612.2(to)-611.8(digest)-613.9(the)-609.2(HMW)]TJ
0 -1.1923 TD
[(gDNA)-218.4(following)-214(the)-219(manufacturers)-219.7(instructions.)-213.7(Library)]TJ
T*
[(was)-341.4(run)-336.2(on)-338.6(a)-338.8(MinION)-337(for)-335.2(72)-165.2(h)-340(with)-339.1(default)-332.7(parameters.)]TJ
0 -1.1869 TD
[(Epi2me)-388.9(was)-390.2(used)-390(to)-389.6(map)-391.1(the)-392.5(resulting)-388.7(FASTQ)-390.4(les)-389(to)]TJ
0 -1.1923 TD
[(the)-511.7(AGG1619)-512.2(plasmid)-510.3(sequence)-510.9(as)-512.1(a)-517.7(reference,)-508.8(with)]TJ
T*
[(30)19.1(8)-291.3(r)0(e)15.8(a)13.4(ds)-285.6(al)18(ig)22.3(n)12.2(i)0(n)16.8(g)12.2(.)-292(F)0(u)16.9(r)13.2(th)16.8(e)13.4(r)-295.7(an)20.3(al)18(y)12.2(s)11(is)-287.8(w)13.5(a)0(s)-284.4(p)0(e)20.3(r)0(f)15.6(o)12.2(r)13.2(m)0(e)19.4(d)-296.7(u)12.2(s)11(in)16.8(g)]TJ
0 -1.1869 TD
[(Gala)-9.3(xy.)]TJ
7.8454 0 0 6.9729 92.8063 204.7181 Tm
(33)Tj
10.4607 0 0 10.4607 105.8456 200.0692 Tm
[(Porec)-8.1(hop)-499.8(\(v0.2.)-8.2(4;)-492.6(http)-9.7(s:/)-6.8(/git)-6.5(hub.)-5.9(com/)-7.1(rrwi)-9.6(ck/)]TJ
-4.4061 -1.1923 TD
[(P)-6.2(orecho)-9.9(p\))-424.3(was)-428.1(used)-428(t)-6.2(o)-421.3(trim)-432.2(adap)-8.3(ters)-429.2(from)-426.8(all)-432.6(FASTQ)]TJ
0 -1.1869 TD
[()-6.2(les.)-365.8(All)-362.1(l)-7(es)-360.3(wer)-8.6(e)-360.5(compi)-11.1(led)-359.9(i)-6.2(nto)-361.1(a)-360.5(sing)-8.6(le)-361.3(F)-6.2(ASTA)-362.5(an)-6.8(d)]TJ
0 -1.1923 TD
[(l)20.1(te)18(re)21.3(d)-291.3(b)0(a)20.3(s)0(e)19.1(d)-291.3(u)12.2(p)0(o)19.1(n)-291.3(al)18(ig)22.3(n)12.2(m)0(e)19.4(n)0(t)-281.2(t)0(o)-281.2(t)0(h)16.8(e)-290.1(5)]TJ
/F10 1 Tf
15.1424 0 TD
()Tj
/F7 1 Tf
.542 0 TD
[(en)20.3(d)-291.3(o)12.2(f)-295.7(t)0(h)22.3(e)-290.1(ho)19.1(m)11.4(o)12.2(l-)]TJ
-15.6844 -1.1923 TD
[(og)19.1(y)-226.2(a)0(r)15.8(m)-227(in)-216.2(th)22.3(e)-230.4(r)13.2(ef)15.8(e)13.4(r)0(e)15.8(n)12.2(ce)-217(s)11(e)0(q)20.3(u)0(e)20.3(n)0(c)20.3(e)-230.5(u)12.2(s)0(i)15.7(n)12.2(g)-226.2(B)10.2(io)22.3(py)19.1(th)22.3(on)19.1(.)]TJ
7.8454 0 0 6.9729 272.6361 154.885 Tm
-.0158 Tc
(34)Tj
10.4607 0 0 10.4607 282.7841 150.2362 Tm
-.0123 Tc
(The)Tj
-21.3208 -1.1869 TD
-.0185 Tc
[(46)-223.1(remainin)4.6(g)-228.5(r).2(e)5.8(a).4(ds)-224.2(wer)5.6(e)-227.3(ma)5.8(nu)4.6(ally)-223(mapp)4.6(ed)-223(to)-223(the)-227.3(c)5.8(on)4.6(stru)4.6(ct/)]TJ
0 -1.1923 TD
0 Tc
[(ge)-6.8(nome)-308.4(interf)-9.1(ace)-8.5(.)]TJ
1.0514 -1.1923 TD
(The)Tj
/F11 1 Tf
2.0703 0 TD
(Pxkmo)Tj
/F7 1 Tf
3.2355 0 TD
[(knockout)-504.7(line)-512.5(was)-509.4(generated)-509.5(previous-)]TJ
-6.3572 -1.1869 TD
(ly.)Tj
7.8454 0 0 6.9729 70.526 105.1086 Tm
(27)Tj
10.4607 0 0 10.4607 81.6945 100.4598 Tm
(The)Tj
/F11 1 Tf
1.8752 0 TD
(Pxyellow)Tj
/F7 1 Tf
3.9943 0 TD
[(knockout)-315(line)-312(was)-314.3(generated)-314.4(follow-)]TJ
-7.9668 -1.1923 TD
[(ing)-312.4(previous)-304.3(protocols,)]TJ
7.8454 0 0 6.9729 157.2661 92.6362 Tm
(35)Tj
10.4607 0 0 10.4607 168.3212 87.9874 Tm
[(except)-310(using)-310.7(a)-311.7(newly)-307.1(designed)]TJ
-10.3786 -1.1923 TD
[(sgRNA)-308.1(\()]TJ
/F11 1 Tf
3.6311 0 TD
-.0003 Tc
(Pxyellow-sgRNA4)Tj
/F7 1 Tf
7.2894 0 TD
0 Tc
[(\))-311.9(targeting)-305(the)-305.7(third)]TJ
/F11 1 Tf
8.2541 0 TD
(Pxyellow)Tj
/F7 1 Tf
-19.1746 -1.1869 TD
(exon.)Tj
/F9 1 Tf
/Cs8 cs 1 scn
24.0035 60.7159 TD
[(Assessment)-300.9(of)-295.3(Cas9)-304(somatic)-298.4(editing)]TJ
0 -1.2411 TD
[(and)-298.4(homing)-296.2(efciency)]TJ
/F7 1 Tf
0 0 0 rg
0 -1.2031 TD
[(To)-381.8(assess)-384.2(somatic)-379(editing)-380.4(efciency,)-386.4(reciprocal)-379.3(crosses)]TJ
0 -1.1869 TD
[(were)-423.4(conducted)-419.6(using)-419.1(Cas9)-424.5(and)-417.3(sgRNA)-421.9(lines.)-424.9(In)-418.9(each)]TJ
0 -1.1923 TD
[(cross)-367.1(replicate,)-366.9(four)-366.3(Cas9)-370.4(heterozygote)-366.6()51.2(grandparents)45.5()]TJ
T*
[(and)-645(four)-642.7(sgRNA)-644.1(heterozygote)-643()45.8(grandparents)50.9()-642.5(were)]TJ
0 -1.1869 TD
[(crossed,)-416.6(with)-420.3(F)]TJ
7.8454 0 0 6.9729 377.9716 621.1274 Tm
(1)Tj
10.4607 0 0 10.4607 386.3054 622.8849 Tm
[(eggs)-417.1(collected)-422.8(and)-422.8(reared)-419.6(until)-422.4(pupa-)]TJ
-7.2135 -1.1923 TD
[(tion.)-296.2(F)]TJ
7.8454 0 0 6.9729 338.6267 608.655 Tm
(1)Tj
10.4607 0 0 10.4607 345.6566 610.4125 Tm
[(pupae)-299.5(were)-298.7(screened)-298.5(for)-297.3(uorescence)-295.3(and)-298.1(body/)]TJ
-3.3276 -1.1923 TD
[(eye)-394.5(pigmentation)-391.5(patterns)-392.1(based)-398.3(on)-392.8(mutant)-391.3(phenotypes)]TJ
0 -1.1869 TD
[(reported)-295.5(previously.)]TJ
7.8454 0 0 6.9729 395.7164 590.1731 Tm
(27,35)Tj
10.4607 0 0 10.4607 321.7889 573.0518 Tm
-.0145 Tc
[(Note)-353.3(that)]TJ
/F11 1 Tf
4.043 0 TD
(Pxyellow)Tj
/F7 1 Tf
3.9401 0 TD
-.015 Tc
[(mosaics)-377.9(a)3.8(re)-375.5(only)-371.3(observable)-375.5(when)]TJ
-9.0291 -1.1923 TD
-.0151 Tc
[(they)-241.3(co-occur)-240.3(in)-235.9(an)-235.9(individual)-238.1(displaying)-241.3(the)-240.1()46.9(stripy)46.9()-234.9(p)-2.9(he-)]TJ
0 -1.1869 TD
-.0159 Tc
[(no)-3.7(type.)-432.5(T)1.9(h)-3.7(i)-.5(s)-427.6(p)-3.7(heno)-3.7(typ)-3.7(e)-430.6(occurs)-433(naturally)-431.8(an)-3.7(d)-426.4(c)-2.5(o)-3.7(n)1.8(sis)-4.8(t)-.5(s)-427.6(o)-3.7(f)]TJ
0 -1.1923 TD
-.0152 Tc
[(two)-214.3(d)2.5(arkly)-214.3(p)-3(igment)-15.2(e)13.7(d)-219.7(areas)-215.5(runni)-15.2(n)12.5(g)-214.3(laterally)-219.8(along)-214.3(t).2(he)-213.1(dor-)]TJ
T*
-.0133 Tc
[(sal)-203.8(s)-2.3(u)4.4(r)-.1(f)5.4(ace)-205.8(of)-206(th)4.4(e)-205.8(p)-1.1(u)4.4(p)-1.1(a)5.6(e).1(.)-207.7(T)4.5(her)5.4(e).1(fo)4.3(re,)-202.3(t)2.1(he)-205.8(nu)4.4(mbe)5.6(r)-206(of)]TJ
/F11 1 Tf
19.2776 0 TD
-.0152 Tc
(Pxyellow)Tj
/F7 1 Tf
-19.2776 -1.1869 TD
[(mosai)-15.3(c)13.6(s)-286.1(was)-280.7(calculated)-284.9(a)-1.9(gainst)-287.1(the)-289.1(number)-283.9(d)2.4(isplaying)-284.9(t).1(his)]TJ
0 -1.1923 TD
-.0165 Tc
[(ph)-4.3(en)-4.3(oty)-4.3(p)1.2(e)-290.3(r)2.2(ath)-4.3(e)2.3(r)-290.5(t)-1.1(han)-291.5(t)-1.1(he)-290.3(total)-288.3(n)1.2(u)-4.3(m).3(ber)-290.5(o)1.2(f)-290.5(pup)-4.3(ae.)-292.2(T)1.3(h)-4.3(e)-284.9(mu)-4.3(-)]TJ
T*
-.0158 Tc
[(tagenesis)-362.4(o)1.9(f)-360.3(t)-.4(arget)-358.1(s).7(i)-15.8(t)9.7(es)-362.4(was)-362.4(c)-2.4(onrmed)-361.3(in)-355.8(a)-360.1(s)-4.8(ubs)-4.8(et)-358.1(of)-360.2(so)-3.6(-)]TJ
0 -1.1869 TD
-.0152 Tc
[(matic)-299.8(mosaics)-296.8(u)-3(sing)-301(a)-299.8(T)-2.8(7)-301(e)3.6(ndonuclease)-299.9(a)-1.8(ssay)-301(\(NEB\).)]TJ
1.046 -1.1923 TD
0 Tc
[(To)-419.7(assess)-422.1()45.8(homing)53.5()-420.3(activity,)-422.7(transheterozygo)5.9(us)-421.1(F)]TJ
7.8454 0 0 6.9729 541.9841 459.2125 Tm
(1)Tj
10.4607 0 0 10.4607 545.8959 460.97 Tm
(s)Tj
-22.4697 -1.1923 TD
[(containing)-434.7(both)-435.6(Cas9)-440.8(and)-433.6(sgRNA)-438.2(transgenes)-435.8(\(parents\))]TJ
0 -1.1869 TD
.0202 Tc
[(were)-481.2(s)4.1(elected)-477(from)-477.8(t)-2.3(h)5.3(e)-481.2(abov)5.3(e)-481.2(c)1.1(ross)-472.8(an)5.3(d)-477(t)-2.3(hemselves)]TJ
0 -1.1923 TD
0 Tc
[(crossed)-254.7(to)]TJ
/F11 1 Tf
4.2869 0 TD
(Pxyellow)Tj
/F7 1 Tf
3.9401 0 TD
(or)Tj
/F11 1 Tf
1.0839 0 TD
(Pxkmo)Tj
/F7 1 Tf
2.9808 0 TD
[(knocko)-6.7(ut)-248.7(lines)-8.3(,)-248.6(depending)]TJ
-12.2917 -1.1869 TD
[(o)12.2(n)-226.2(wh)20.3(ic)18(h)-226.2(s)11(g)12.2(R)10.2(NA)-217(li)20(n)12.2(e)-230.5(w)13.5(a)0(s)-219.4(u)12.2(s)11(e)0(d)-218.2(i)0(n)-216.2(t)0(h)22.3(e)-225(in)16.8(it)20(ia)23.4(l)-228.4(c)0(r)21.3(o)0(s)17.9(s)-227.4(\()13.2(F)0(i)20.1(g)12.2(.)-226.9(3)0(\))20.1(.)]TJ
0 -1.1923 TD
[(I)13.2(n)-237.1(ea)16(ch)-229(c)13.4(r)0(o)20.1(s)0(s)-227.2(r)0(e)15.8(p)12.2(li)20(ca)16(te)23.5(,)-237.8(a)-241.3(m)11.4(i)0(n)22.3(i)0(m)21.4(u)0(m)-231.1(o)12.2(f)-236.1(t)0(h)16.8(r)13.2(ee)-227.9(tr)17.8(an)20.3(s)11(h)12.2(et)18(er)15.8(o)12.2(z)0(y)20.3(-)]TJ
T*
[(g)12.2(o)12.2(t)0(e)18(s)-465.9(a)13.4(n)0(d)-452.5(t)0(h)16.8(r)13.2(ee)-455.5(k)12.2(n)12.2(o)0(c)20.2(k)0(o)19.1(u)12.2(t)-466.9(i)0(n)22.3(d)12.2(iv)16.8(id)22.3(u)12.2(a)0(l)18(s)-460.5(we)16.1(r)13.2(e)-463.5(re)15.8(c)13.4(i)0(p)16.8(r)13.2(oc)20.2(al)18(ly)]TJ
0 -1.1869 TD
[(c)13.4(r)0(o)20.1(s)0(s)16.7(e)0(d)20.3(,)-281.1(wi)18.1(th)-270.4(v)22.3(e)-284.7(r)13.2(ep)20.3(li)20(ca)16(te)18(s)-281.6(c)13.4(on)19.1(d)12.2(u)0(c)20.2(t)0(e)18(d)-280.4(fo)20.1(r)-284.8(e)0(a)21.4(c)0(h)-272.4(c)0(o)20.3(m)0(b)18.2(i)0(n)22.3(a)0(-)]TJ
0 -1.1923 TD
[(ti)20(o)12.2(n)12.2(.)-275.7(An)-267(ex)20.3(ce)16(p)12.2(t)0(i)20(o)12.2(n)-275(t)0(o)-265(t)0(h)16.8(e)-273.8(ab)20.3(ov)19.1(e)-273.8(p)0(r)20.1(o)0(c)20.2(e)0(s)19.1(s)-276.2(wa)16.1(s)-270.8(m)0(a)19.4(d)0(e)-267(f)0(o)20.1(r)-274(th)16.8(e)]TJ
T*
[(1)12.2(9)12.2(0)0(6)-447(l)0(i)20(n)12.2(es)-447(wh)20.3(er)15.8(e)13.4(,)-454.5(du)19.1(e)-458.1(t)0(o)-443.8(t)0(h)22.3(e)-458.1(la)18(r)13.2(g)0(e)-445.8(n)0(u)19.1(m)11.4(be)20.3(r)-458.3(o)12.2(f)-458.3(l)0(i)20(n)12.2(es)-447(to)]TJ
0 -1.1869 TD
[(a)13.4(s)0(s)16.7(e)0(s)19.1(s)11(,)-292(t)0(h)22.3(e)-290.1()56.6()13.2(gr)20.1(an)20.3(dp)19.1(ar)21.3(en)20.3(t)66.6()-290.3(f)0(a)21.3(c)0(t)18(o)12.2(r)-290.3(w)0(a)16(s)-287(n)12.2(o)0(t)-281.2(a)0(p)20.3(p)0(l)22.3(i)0(e)18(d)0(.)]TJ
1.046 -1.1923 TD
[(In)-283.4(all)-286.3(cases,)-289.2(the)-284.1(third)-283.7(instar)-287.7(larvae)-287.3(\()]TJ
/F11 1 Tf
14.3403 0 TD
(Pxyellow)Tj
/F7 1 Tf
3.9672 0 TD
[(cross\))-288.8(or)]TJ
-19.3534 -1.1923 TD
[(pupae)-418.8(\()]TJ
/F11 1 Tf
3.1434 0 TD
(Pxkmo)Tj
/F7 1 Tf
3.1488 0 TD
[(cross\))-424.3(of)-418.9(F)]TJ
7.8454 0 0 6.9729 424.9133 309.77 Tm
(2)Tj
10.4607 0 0 10.4607 433.1904 311.5275 Tm
[(progeny)-421.6(were)-417.9(screened)-423.2(for)]TJ
-11.6955 -1.1869 TD
[(uorescence)-257.4(\(to)-262.6(assess)-264.9(gene)-257.6(drive)-258.6(activity\))-263.8(and)-260.2(knockout)]TJ
0 -1.1923 TD
[(phenotypes)-323(\(to)-322.2(assess)-330(germline/som)-10.9(atic)-321.6(edi)-7.7(ting)-324(efcien-)]TJ
T*
[(cy\).)-583.6(Control)-586.4(crosses)-586.5(were)-580.5(conducte)-9.1(d)-578.5(b)0(y)-582.5(recip)-8.1(rocally)]TJ
0 -1.1869 TD
[(crossing)-321.8(ea)16(ch)-299.5(s)11(g)12.2(R)10.2(N)0(A)-298.3(l)0(i)20(n)0(e)-299.5(a)0(s)-300.7(h)12.2(et)18(er)21.3(oz)20.2(yg)19.1(o)12.2(t)0(e)18(s)-308.7(to)-297.5(it)20(s)-308.7(r)0(e)15.8(s)11(p)12.2(e)0(c)16(-)]TJ
0 -1.1923 TD
[(ti)20(v)12.2(e)-290.1(k)12.2(n)0(o)19.1(c)0(k)20.3(o)12.2(ut)-281.2(li)20(n)12.2(e)-290.1(an)20.3(d)-291.3(s)11(co)20.3(ri)17.8(n)12.2(g)-291.3(th)22.3(e)-290.1(s)0(a)19.1(m)11.4(e)-290.1(d)0(e)20.3(t)0(a)18(i)0(l)20(s)-292.5(a)13.4(s)-292.5(ab)20.3(o)12.2(v)0(e)20.3(.)]TJ
/F9 1 Tf
/Cs8 cs 1 scn
0 -2.3142 TD
[(Statistical)-301.5(analyses)]TJ
/F7 1 Tf
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[(Analyses)-352.9(of)-348.5(sgRNA)-356.9(cassette)-353.9(inheritance)-354(was)-352.3(conducted)]TJ
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[(using)-370.4(R)-380(v3.6.2)-372.4(\(The)-371.4(R)-374.6(Foundation)-376.2(for)-373.2(Statistical)-377.1(Com-)]TJ
0 -1.1923 TD
(puting\),)Tj
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(36)Tj
10.4607 0 0 10.4607 356.7684 187.5968 Tm
[(and)-493.2(gures)-496.8(were)-499.2(generated)-493.3(using)-495(ggplot2)]TJ
7.8454 0 0 6.9729 542.0975 192.2456 Tm
(37)Tj
10.4607 0 0 10.4607 310.8472 175.1811 Tm
[(and)-330.6(patchwork.)]TJ
7.8454 0 0 6.9729 376.2708 179.8298 Tm
(38)Tj
10.4607 0 0 10.4607 387.6093 175.1811 Tm
[(The)-330.4(proportion)-329.8(of)-337.6(offspring)-329.2(inheriting)]TJ
-7.3382 -1.1923 TD
[(th)22.3(e)-295.5(s)11(g)12.2(R)10.2(NA)-287.4(ca)21.4(s)11(s)0(e)19.1(t)0(t)20(e)-295.5(wa)16.1(s)-292.5(a)0(n)20.3(a)0(l)18(y)12.2(ze)21.4(d)-296.7(u)12.2(s)11(i)0(n)16.8(g)-291.3(a)-295.5(b)12.2(in)22.3(om)18.2(ia)18(l)-293.5(G)13.5(LM)]TJ
T*
.0097 Tc
[(\()44.6(logit)50()-426.9(link\))-426.9(with)-427.9(control)-430.1(c)1.4(ross)-429.1(outcomes)-423.7(s)-.9(et)-424.7(as)-429.1(the)]TJ
0 -1.1869 TD
[(intercept.)-358.1(Approximate)-361.7(95%)-355(condence)-356.2(i)-2(ntervals)-358.6(were)]TJ
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[(calculated)-622.9(using)-628.3(t)3.6(he)-627.1(Sidak)-628.3(a)1.6(djustment)-630.5(f)1.4(or)-627.3(multiple)]TJ
T*
.0091 Tc
[(compar)-4.8(isons.)]TJ
1.046 -1.1869 TD
0 Tc
[(Figures)-340.5(of)-337.6(germline)-337.5(cleavage)-342.7(percentages)-338.7(were)-342.1(made)]TJ
-1.046 -1.1923 TD
.0192 Tc
[(using)-385.9(G).1(ra)-5.3(phPad)-385.9(s)-2.3(oftw)-5.3(are.)-386.6(D)-5.3(i)2.1(ffe)-5.3(rences)-392.5(were)-390.1(a).1(nalyz)-5.3(e).1(d)]TJ
T*
.021 Tc
[(with)-373.2(one-way)-373.2(analysis)-374.4(of)-372.2(variance)-372(or)-377.6(an)-373.2(in)6.1(dep)6.1(e)-3.5(n)6.1(d).7(en)6.1(t)]TJ
/F11 1 Tf
0 -1.1869 TD
0 Tc
(t)Tj
/F7 1 Tf
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[(-t)-3.5(est)-301.6(u)-1.3(si)-3.5(ng)-299.4(SP)-3.5(SS)-301.6(S)-3.5(t)1.9(at)-3.5(ist)-3.5(i)1.9(cs)-306(22.0)-299.4(\(IB)-3.3(M)-299.2(C)-3.3(orp\).)]TJ
/F5 1 Tf
9.9626 0 0 9.9626 59.7543 753.8455 Tm
0 Tc
[(G)-5.9(ENE)-299.6(DRI)-8.2(VE)-302.2(IN)]TJ
/F14 1 Tf
7.0278 0 TD
[(PLUTE)-8.4(LLA)-305.9(XYLOST)-8.9(ELLA)]TJ
/F5 1 Tf
41.6207 0 TD
(3)Tj
ET
endstream
endobj
71 0 obj
<>stream
/GS1 gs
BT
/F9 1 Tf
10.4607 0 0 10.4607 62.022 698.2297 Tm
/Cs8 cs 1 scn
0 Tc
0 Tw
[(Western)-300.1(blot)]TJ
/F7 1 Tf
0 0 0 rg
0 -1.2032 TD
[(Moth)-234.9(carcass,)-235.4(ovary,)-234(and)-233.1(testis)-237.9(samples)-237.1(from)-231.7(2-)-234.7(to)-237.9(3-day-)]TJ
0 -1.1923 TD
[(old)-214.8(adults)-212.8(were)-217.4(homogenized)-211.7(on)-214(ice)-217.8(in)-216.2(100)]TJ
/F13 1 Tf
17.1477 0 TD
(l)Tj
/F7 1 Tf
.5528 0 TD
.2152 Tc
(L1)Tj
/F10 1 Tf
1.4958 0 TD
0 Tc
()Tj
/F7 1 Tf
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(passive)Tj
-19.9117 -1.1869 TD
.021 Tc
[(ly)6.1(sis)-461.1(b)6.1(uffer)-458.9(\()1.7(PLB)4.1(\))-458.9(inclu)6.1(d).7(ing)-459.9(Halt)-456.7(P)-1.5(rot)3.9(e)-3.5(ase)-458.8(I)1.7(nhi)3.9(bit)3.9(o).7(r)]TJ
0 -1.1923 TD
0 Tc
[(Cocktail)-331.1(\(Thermo)-323.6(Fis)-6.8(her)-324.2(Scientic\).)-327.8(After)-326.8(15)-170.6(min)-318.6(of)-326.8(in-)]TJ
T*
[(cubatio)-8.5(n)-285.8(a)0(t)-290.9(room)-292.3(temperatu)-9.7(re,)-287(sample)-9.7(s)-287(were)-293.3(centrifug)-9.1(ed)]TJ
0 -1.1869 TD
[(for)-232.3(10)-165.2(min)-231.9(at)-236.7(10,000)]TJ
/F11 1 Tf
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(g)Tj
/F7 1 Tf
.7316 0 TD
[(and)-227.7(4)]TJ
/F10 1 Tf
2.1787 0 TD
()Tj
/F7 1 Tf
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[(C.)-233(An)-229(additional)-236.4(50)]TJ
/F13 1 Tf
8.021 0 TD
(l)Tj
/F7 1 Tf
.5528 0 TD
[(L)-226(PLB)]TJ
/F10 1 Tf
-20.1826 -1.1923 TD
(+)Tj
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[(protease)-318.2(inhibitor)-315(was)-314.3(added)-310.4(to)-313.8(the)-311.2(samples)-313(and)-314.4(incu-)]TJ
-.8617 -1.1923 TD
[(bated)-307.2(for)-302.7(a)-300.9(further)-302.5(15)-170.6(min)-302.3(at)-301.7(room)-303.1(temperature.)]TJ
1.046 -1.1869 TD
[(Protein)-303.5(concentrations)-307.6(were)-304.1(determined)-306.4(using)-299.9(the)-305.7(DC)]TJ
-1.046 -1.1923 TD
[(Protein)-341.5(Assay)-345.2(\(Bio-Rad\))-341.7(using)-337.8(a)-344.3(bovine)-339.5(serum)-345.1(albumin)]TJ
T*
[(standard)-346.5(curve.)-346.6(Absorbance)-349.9(values)-344.9(were)-352.9(obtained)-343.1(using)]TJ
0 -1.1869 TD
[(a)-300.9(microplate)-297.8(reader)-300.3(\(GloMax\))-300.7(at)-301.7(750)-163.8(nm.)]TJ
1.046 -1.1923 TD
[(For)-202.9(each)-207.6(sample,)-204.1(equal)-204.2(amounts)-199.2(of)-202.1(total)-204.4(protein)-203.8(\(3)20.1(5)]TJ
/F13 1 Tf
20.4482 0 TD
(l)Tj
/F7 1 Tf
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(g\))Tj
-22.0361 -1.1923 TD
0 Tc
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0 -1.1869 TD
[(an)20.3(d)-291.3(d)12.2(en)20.3(at)18(u)12.2(r)0(e)21.3(d)-291.3(fo)20.1(r)-290.3(5)-155.8(m)11.4(i)0(n)-281.2(a)0(t)-280(9)12.2(5)]TJ
/F10 1 Tf
11.7931 0 TD
()Tj
/F7 1 Tf
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-.0102 Tc
(C.)Tj
-11.1319 -1.1923 TD
0 Tc
[(Samples)-373.6(were)-369.2(loaded)-370.8(onto)-370.6(420%)-370(precast)-368.4(polyacryl-)]TJ
-1.046 -1.1923 TD
[(amide)-405.5(gels)-403.1(\(Bio-Rad\))-406.7(and)-406.5(run)-401.3(at)-404.7(200)-163.8(V.)-408.6(A)-403.9(chemilumi-)]TJ
0 -1.1869 TD
[(nesce)20.2(nt)-330(p)12.2(r)0(o)20.1(t)0(e)18(i)0(n)-324.6(l)0(a)18(d)12.2(de)20.3(r)-339(\()0(8)20.1()0(2)19.1(6)12.2(0)-155.8(k)0(D)20.3(a)0(;)-328.8(L)0(I)20.3(-)0(C)18.1(O)13.5(R)10.2(\))-339(wa)16.1(s)-335.8(l)0(o)22.3(a)0(d)14.8(e)13.4(d)]TJ
0 -1.1923 TD
[(al)18(o)12.2(n)12.2(g)0(s)17.9(i)0(d)22.3(e)-338.8(th)16.8(e)-333.4(p)0(r)20.1(o)0(t)16.8(e)13.4(in)-330(s)11(a)0(m)19.4(p)12.2(le)18(s)-335.8(t)0(o)-330(e)0(s)19.1(t)0(i)20(m)11.4(at)18(e)-338.8(t)0(h)22.3(e)-338.8(p)12.2(o)0(s)17.9(i)0(t)20(i)0(o)22.3(n)-340(o)12.2(f)]TJ
T*
[(the)-430.4(158)-169.2(kDa)-427(Cas)-8.6(9)-426.8(\(FLAG-tagged\))-438.8(band.)-431.5(Proteins)-433.4(were)]TJ
0 -1.1869 TD
[(transf)-8.1(erred)-449.3(onto)-451.9(a)-447.2(nitro)-7.3(cellulose)-452.3(membran)-10.1(e)-447.2(\(Bio-Rad\).)]TJ
0 -1.1923 TD
[(Af)21.3(te)18(r)-246.9(t)0(r)17.8(a)0(n)20.3(s)11(fe)21.3(r,)-240.8(u)12.2(n)12.2(s)0(p)17.9(e)0(c)21.4(i)0()20.1(c)-252.1(b)12.2(i)0(n)22.3(d)0(i)16.8(n)12.2(g)-247.9(s)11(it)20(es)-241.1(w)13.4(e)0(r)15.8(e)-246.7(b)12.2(l)0(o)16.8(c)13.4(ke)20.3(d)-247.9(o)0(v)19.1(e)0(r)21.3(-)]TJ
T*
[(night)-463.5(at)-464.3(4)]TJ
/F10 1 Tf
4.2165 0 TD
()Tj
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[(C)-461.3(with)-463.7(3%)-460.9(milk)-465.7(powder)-465.1(\()-84.3(Marvel\))-464.7(in)-465.5(Tris-)]TJ
-4.6175 -1.1869 TD
[(buffered)-330.2(salin)-8.3(e)-322.6(solutio)-8.9(n)-323.8(\(Bio-Rad\))-330.8(inclu)-7.1(ding)-327.2(1%)-325.4(Tween)]TJ
0 -1.1923 TD
[(20)-376.6(\(TBS-Tween;)-380.6(SigmaAld)-8.7(rich\).)-376.1(Anti-FLAG-ta)-12.1(g)-372.6(anti-)]TJ
T*
[(body)-503.8(\(Abcam\))-505.7(was)-498.6(dilute)-7.9(d)-497.2(1:1,0)-6.8(00)-495.8(in)-503.4(3%)-498.8(milk/TBS-)]TJ
0 -1.1869 TD
[(Tween,)-384.1(and)-379.4(the)-381.6(membrane)-386.9(incubated)-383.9(overn)-7.1(ight)-378.2(at)-383(4)]TJ
/F10 1 Tf
21.543 0 TD
()Tj
/F7 1 Tf
.3956 0 TD
(C.)Tj
-21.9386 -1.1923 TD
[(Membranes)-364(were)-352.9(de)-6.8(veloped)-358.6(using)-354.1(Pierce)-359.9(ECL)-355.1(Western)]TJ
T*
[(Blott)-7.1(ing)-437(Substr)-7(ate)-440(\(The)-8.3(rmo)-440(Fisher)-444.8(Scienti)-8.3(c\),)-438.7(scann)-8.1(ed)]TJ
0 -1.1869 TD
[(using)-354.1(a)-355.1(Gel)-353.3(Doc)-356.5(imager)-354.3(\(Bio-Ra)-9.5(d\),)-353.2(and)-352.3(analy)-9.1(zed)-351.1(using)]TJ
0 -1.1923 TD
[(Image)-304.8(Lab)-303.3(software)-306.3(\(Bio-Ra)-9.5(d\).)]TJ
/F5 1 Tf
/Cs8 cs 1 scn
0 -2.3142 TD
(Results)Tj
/F9 1 Tf
0 -1.2357 TD
[(Cas9)-298.5(line)-300.1(generation)-301.5(and)-298.4(characterization)]TJ
/F7 1 Tf
0 0 0 rg
0 -1.2032 TD
[(Previously,)-457(we)-460.9(showed)-456.5(that)]TJ
/F11 1 Tf
12.0857 0 TD
(Pxvasa)Tj
/F7 1 Tf
3.3493 0 TD
[(was)-460.7(preferentially)]TJ
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[(expressed)-445.8(in)-449.2(adult)-446.1(soma)-445(and)-444.4(gonads,)-446.1(and)-444.4(the)-446.6(selected)]TJ
/F11 1 Tf
0 -1.1869 TD
(Pxvasa)Tj
/F7 1 Tf
3.2518 0 TD
[(promoter)-351.7(fragment)-356(drove)-353.9(Cas9)-354.1(somatic)-357.4(and)-357.7(go-)]TJ
-3.2518 -1.1923 TD
[(nadal)-312.6(transcription)-312.6(in)-319.2(a)-311.7(single)-315.8(generated)-314.4(line)-312(\(1536A\).)]TJ
7.8454 0 0 6.9729 293.2723 229.6062 Tm
(26)Tj
10.4607 0 0 10.4607 62.022 212.485 Tm
[(He)21.5(re)15.8(,)-329.9(t)0(h)22.3(r)0(e)15.8(e)-328(ad)20.3(di)16.8(ti)20(o)12.2(n)12.2(a)0(l)-323.4(1)12.2(53)19.1(6)-329.2(l)0(i)20(n)0(e)20.3(s)-335.8(w)13.4(e)0(r)15.8(e)-328(de)20.3(ve)20.3(lo)16.8(p)12.2(e)0(d)-321.2(\()0(F)17.9(i)0(g)22.3(.)-329.9(1)]TJ
0 -1.1869 TD
[(an)20.3(d)-589.3(T)12.4(ab)20.3(le)-578.1(1\))20.1(.)-590(Moreover,)-606.7(homologs)-600(of)-597.8(nine)-602.4(putat)-7.1(ive)]TJ
0 -1.1923 TD
[(germline)-250.8(expressing)-247.3(genes)-246.5(were)-244.5(ident)-7.1(ied)-241.5(in)-243.3(DBM)-245.7(\(Sup-)]TJ
0 -1.1869 TD
[(plementar)-9.7(y)-508(Table)-518.3(S2\).)-516.6(RT-PCR)-513.5(showed)-516.1(that)]TJ
/F11 1 Tf
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(Pxbgcn)Tj
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(,)Tj
/F11 1 Tf
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(Pxsds3)Tj
/F7 1 Tf
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/F11 1 Tf
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(Pxshu1)Tj
/F7 1 Tf
2.9483 0 TD
(,)Tj
/F11 1 Tf
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(Pxshu2)Tj
/F7 1 Tf
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(,)Tj
/F11 1 Tf
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(Pxpiwi)Tj
/F7 1 Tf
2.7965 0 TD
[(,)-465.4(and)]TJ
/F11 1 Tf
2.6339 0 TD
(PxnanosN)Tj
/F7 1 Tf
4.585 0 TD
(were)Tj
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[(ubiquit)-7.7(ously)-207.8(transcribed)-212.2(in)-205.4(embry)-7.9(os,)-203.6(larva)-8.1(e,)-202.7(an)-6.8(d)-204.6(adult)-207.6(tis-)]TJ
0 -1.1869 TD
[(sues,)-303.8(whereas)]TJ
/F11 1 Tf
5.8532 0 TD
[(Pxnan)-7.9(osO)]TJ
/F7 1 Tf
4.4712 0 TD
(and)Tj
/F11 1 Tf
1.7505 0 TD
(PxnanosM)Tj
/F7 1 Tf
4.585 0 TD
[(were)-298.7(ex)-6.8(pressed)]TJ
-16.6599 -1.1923 TD
[(in)-286.7(female)-288.1(ovaries)-288.3(and)-281.8(early)-289.9(embryos.)-287.4(In)-283.4(contrast,)-293.8(detect-)]TJ
T*
(able)Tj
/F11 1 Tf
1.8969 0 TD
(Pxmeiw68)Tj
/F7 1 Tf
4.4007 0 TD
[(expressio)-10.3(n)-215.4(was)-222.2(restricte)-11.3(d)-220.8(t)0(o)-216.2(adult)-223.9(gonads)-7.8(,)]TJ
-6.2976 -1.1869 TD
(and)Tj
/F11 1 Tf
1.7831 0 TD
(PxnanosP)Tj
/F7 1 Tf
4.3953 0 TD
[(was)-336(preferen)-9.3(tially)-335.3(detect)-8.7(ed)-332(in)-335.4(adults)-332(\(es-)]TJ
-6.1784 -1.1923 TD
[(pecial)-8.7(ly)-275.8(female)-282.7(ovaries;)-283.7(Suppleme)-7.9(ntary)-280.3(Fig.)-275.9(S1A\),)-280.9(mak-)]TJ
T*
[(ing)-474.9(them)-478.6(better)-480.4(candid)-7.7(ates)-477.8(for)-476.1(further)-481.4(constru)-7.7(ction)-478.6(of)]TJ
0 -1.1869 TD
[(Cas9)-213.2(lines)-219.6(for)-210.6(homing)-216.8(drive)-215.2(generation.)-218.2(The)-211.2(putat)-7.1(ive)-213.6(pro-)]TJ
23.998 28.4909 TD
[(mote)-7.1(rs)-393(and)-390.2(5)]TJ
/F10 1 Tf
5.4684 0 TD
()Tj
/F7 1 Tf
.2493 0 TD
(/3)Tj
/F10 1 Tf
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()Tj
/F7 1 Tf
.6395 0 TD
[(regu)-8.5(latory)-394.9(regions)-392.5(of)-391.8(thes)-7.5(e)-393(two)-392.4(genes)]TJ
-7.1376 -1.1869 TD
[(wer)-8.6(e)-317.2(cloned)-322(to)-319.2(direct)-323.2(Cas9)-321.6(expressio)-10.3(n,)-317.7(with)-322.8(seven)-322.4(1906)]TJ
0 -1.1923 TD
(\()Tj
/F11 1 Tf
.336 0 TD
[(Pxmeiw68-Cas)-12.9(9)]TJ
/F7 1 Tf
6.5686 0 TD
[(\))-333.6(and)-330.6(ve)-332.8(2093)-330.4(\()]TJ
/F11 1 Tf
6.9371 0 TD
[(Pxnan)-7.9(osP-Cas9)]TJ
/F7 1 Tf
6.4548 0 TD
[(\))-333.6(lines)]TJ
-20.2964 -1.1923 TD
[(subs)-7.6(equently)-462.3(gene)-8.3(rated)-463.3(\(Table)-467.2(1\).)-461.6(Flanking)-465.7(PCR)-462.7(con-)]TJ
0 -1.1869 TD
[(rmed)-346.1(indepe)-7.7(ndent)-340.9(inse)-7.5(rtion)-343.3(sites)-345.5(of)-343(each)-343.1(line)-344.5(\(Supple-)]TJ
0 -1.1923 TD
[(ment)-7.1(ary)-356.7(Table)-361.2(S3\).)-359.4(Paired)-361.1(expression)-361.1(pro)-6.4(ling)-356.5(of)-359.3(Cas9)]TJ
T*
[(an)-6.8(d)-426.8(endogeno)-8.1(us)-426.5(ge)-6.8(nes)-429.4(\()]TJ
/F11 1 Tf
10.1455 0 TD
(Pxmeiw68)Tj
/F7 1 Tf
4.1785 0 TD
(,)Tj
/F11 1 Tf
.6829 0 TD
(Pxvasa)Tj
/F7 1 Tf
2.8941 0 TD
[(,)-432.9(and)]TJ
/F11 1 Tf
2.5635 0 TD
(Pxna-)Tj
-20.4645 -1.1869 TD
(nosP)Tj
/F7 1 Tf
2.0053 0 TD
[(\))-311.9(showed)-315.5(that)-312(Cas9)-316.2(mRNA)-309.1(was)-314.3(transcr)-10.9(ibed)-309.8(in)-313.8(both)]TJ
-2.0053 -1.1923 TD
[(t)-6.2(estes)-331.2(and)-330.6(ovaries)-337.1(of)-332.2(all)-329.6(1536,)-335.1(2093,)-329.7(and)-330.6(1906)-330.4(lines)-333.4(ex-)]TJ
T*
[(cept)-308.6(1906B)-303.9(\(Supple)-8.7(mentary)-304.2(Fig.)-308.4(S1B\).)-305.8(Unexpectedly,)-311.5(it)]TJ
0 -1.1869 TD
[(was)-395.6(also)-397.7(observed)-398.5(that)-398.7(expression)-399.1(levels)-395.9(of)-397.3(Cas9)-392(were)]TJ
0 -1.1923 TD
[(much)-205.4(higher)-208.4(in)-205.4(adult)-202.2(car)-8.7(casses)-208.4(than)-201.4(in)-205.4(gonadsthe)-207.8(oppo-)]TJ
T*
[(sit)-6.8(e)-344.3(trend)-345.3(for)-346.1(the)-343.7(respectiv)-10.1(e)-338.8(e)0(n)-6.8(dogenous)-346.5(genes)-344.1(\(Supple)-8.7(-)]TJ
0 -1.1869 TD
[(ment)-7.1(ary)-302.5(Fig.)-303(S1B\).)]TJ
/F9 1 Tf
/Cs8 cs 1 scn
0 -2.2925 TD
[(sgRNA)-302.4(line)-300.1(generation)-296.1(and)-298.4(characterization)]TJ
/F7 1 Tf
0 0 0 rg
0 -1.2032 TD
[(Homozygous)-251.1(null)-253.6(mutations)-249.6(in)]TJ
/F11 1 Tf
12.6331 0 TD
(Pxyellow)Tj
/F7 1 Tf
3.9346 0 TD
(or)Tj
/F11 1 Tf
1.0893 0 TD
(Pxkmo)Tj
/F7 1 Tf
2.9754 0 TD
(cause)Tj
-20.6325 -1.1923 TD
[(visible)-408.7(phenotypes)-404.3(in)-411.3(the)-403.3(body)-406.3(or)-408.1(eye)-410.7(pigmentation)-402.4(of)]TJ
T*
[(DBM,)-418.4(respectively,)-412.7(making)-416.2(them)-413.6(scorable)-415.8(markers)-415.9(for)]TJ
0 -1.1869 TD
[(genome)-309.8(editing)-304.6(events.)]TJ
7.8454 0 0 6.9729 411.307 129.9968 Tm
(27,35)Tj
10.4607 0 0 10.4607 432.1133 125.348 Tm
[(As)-311.5(such)-303.3(we)-309.1(chose)-306.1(these)-308.4(two)]TJ
-11.3812 -1.1923 TD
[(genes)-452.5(as)-452.4(targets)-453.1(for)-449(building)-450.7(sgRNA)-449(expression)-447.9(lines.)]TJ
T*
[(Alt)11.8(ho)13.6(ugh)-304.7(si)10.2(x)]TJ
/F11 1 Tf
5.528 0 TD
[(Px)9.6(U6)]TJ
/F7 1 Tf
2.5743 0 TD
[(pro)16(mot)12(er)10.4(s)-314.1(\()]TJ
/F11 1 Tf
4.6609 0 TD
[(Px)9.6(U6:)17.3(1)]TJ
/F7 1 Tf
3.3981 0 TD
(to)Tj
/F11 1 Tf
1.0893 0 TD
[(Px)9.6(U6:)11.9(6)]TJ
/F7 1 Tf
3.0838 0 TD
[(\))-311.9(hav)16.2(e)]TJ
-20.3344 -1.1869 TD
-.0209 Tc
[(been)-328.4(identi)-5.5(ed)-333.8(a)-2(nd)-333.8(characteri)-5.5(zed)-333.8(i)0(n)-333.8(D)-2(BM)-328.2(somati)-5.5(c)-332.6(c)-2(el)-5.5(ls,)]TJ
7.8454 0 0 6.9729 544.5353 92.6362 Tm
-.0231 Tc
(32)Tj
10.4607 0 0 10.4607 313.0582 75.5149 Tm
-.0208 Tc
[(no)-642.7(informat)-5.4(ion)-642.7(i).1(s)-643.9(a)3.5(vailabl)-5.4(e)-641.5(o)2.3(n)-642.7(t).1(heir)-641.7(germ-cell)-644.9(a)-1.9(ct)-5.4(ivi-)]TJ
0 -1.1923 TD
-.0212 Tc
[(t)-5.8(i)-.3(es.)-562.5(T)-3.4(herefo)-3.5(re,)-567.9(w)3.1(e)-566(e)-2.4(mpl)-5.8(o)1.9(y)-3.5(e)3.1(d)-567.2(a)-560.6(con)-3.5(s).7(ervati)-5.8(ve)-566(ap)-3.5(pro)-3.5(a)3.1(ch)-3.5(,)]TJ
ET
q
1 i
313.625 705.316 m
551.565 705.316 l
551.565 513.751 l
313.625 513.751 l
W n
q
238.023 0 0 191.565 313.625 513.751 cm
/Im2 Do
Q
Q
BT
/F5 1 Tf
9.9626 0 0 9.9626 313.0582 495.8361 Tm
0 Tc
[(FI)-7.1(G.)-336.3(1.)]TJ
/F9 1 Tf
3.8753 0 TD
[(Constr)-10.4(uction)-304.3(of)-306.1(transgeni)-12.1(c)-298.5(lines)-7(.)]TJ
/F5 1 Tf
13.8565 0 TD
(\(A\))Tj
/F9 1 Tf
1.5763 0 TD
(Donor)Tj
-19.308 -1.3031 TD
[(ca)-7.2(ssettes)-306.5(for)]TJ
/F8 1 Tf
5.5312 0 TD
(piggyBac)Tj
/F9 1 Tf
3.6704 0 TD
[(-medi)-9.5(ated)-305.1(transform)-11.7(ation)-306.8(of)-300.4(Cas9)]TJ
-9.2016 -1.2974 TD
[(lines)-7(.)]TJ
/F5 1 Tf
2.4128 0 TD
(\(B\))Tj
/F9 1 Tf
1.5194 0 TD
[(Homol)-10.7(ogy-direct)-11.5(ed)-303.7(repair)-306.1(\(HDR\)-b)-8.3(ased)]TJ
-3.9322 -1.3031 TD
[(inte)-7.4(grati)-8(on)-301.2(of)-300.4(hom)-8.8(ing)-300.2(elements)-311.7(into)-302.2(endo)-7.4(genous)]TJ
0 -1.2974 TD
[(ma)-8.2(rker)-305.7(genes)]TJ
/F8 1 Tf
6.0377 0 TD
[(P)-6.5(xyellow)]TJ
/F9 1 Tf
3.7899 0 TD
(and)Tj
/F8 1 Tf
1.895 0 TD
(Pxkmo)Tj
/F9 1 Tf
2.7144 0 TD
[(.)-303.8(Note)-302.7(that)-305.3(the)-304.7(same)]TJ
-14.4369 -1.3031 TD
[(si)-7.6(ngle-guide)-306.5(RNA)-303.1(\(s)-6.9(gRNA\))-306.6(was)-303.3(express)-9.1(ed)-303.7(by)-301.6(three)]TJ
0 -1.2974 TD
[(d)-7.1(ifferent)-304.9(U6)-307.5(promoter)-11.9(s)-299.2(i)0(n)-300.2(eac)-8(h)-301.6(sgRNA)-306.4(donor)-304.5(con)-7.8(struct.)]TJ
/F8 1 Tf
0 -1.3031 TD
[(Red)-305.5(arro)-9(w:)]TJ
/F9 1 Tf
4.6719 0 TD
[(posi)-8.6(tion)-302.2(of)-306.1(sgRNA)-306.4(target;)]TJ
/F8 1 Tf
10.8633 0 TD
[(bl)-7.5(ack)-301.4(arro)-9(w:)]TJ
/F9 1 Tf
-15.5352 -1.2974 TD
[(ge)-6.4(notyping)-307.8(primer)-9(s.)-301.4(Co)-8(lor)-302.2(imag)-6.8(es)-300(are)-303.7(available)-307(onlin)-8.2(e.)]TJ
/F5 1 Tf
-25.1978 36.3512 TD
(4)Tj
44.8301 0 TD
[(XU)-306.7(ET)-299.4(AL.)]TJ
ET
endstream
endobj
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