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[(2000)-216.6(\(Invitro)9.8(gen\))-219.8(or)-223.8(Dhar)9(mafect)-223.1(\(Dharma)9.2(con\))-219.8(a)-8.3(c)-13.7(c)-8.3(o)-9.4(r)-8.4(d)-9.4(i)-11.7(n)-9.4(g)]TJ
T*
[(t)-6.2(o)-730.2(t)-11.7(h)-9.4(e)-723.6(m)-10.3(a)-13.7(n)-9.4(u)-9.4(f)-8.4(a)-8.2(c)-13.7(t)-6.2(u)-9.4(r)-13.9(e)-8.2(r)-8.4(s)-10.6()-729.2(i)-11.7(n)-9.4(s)-10.6(t)-6.2(r)-13.9(u)-9.4(c)-8.3(t)-11.7(i)-6.2(o)-9.4(n)-9.4(s)-10.6(.)-730.9(Puromycin)-722.1(was)]TJ
0 -1.1869 TD
[(added)-321.2(24)-170.6(h)-318.4(post)-323(transfection)-326.9(at)-323.4(concentratio)-11.2(ns)-318.1(from)-323.8(1)-318.4(t)0(o)]TJ
0 -1.1923 TD
(10)Tj
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(l)Tj
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[(g/mL)-593.4(an)-6.8(d)-589.3(repla)-8.1(ced)-595(daily)-597.8(until)-595.8(all)-595.2(control)-599.4(green)]TJ
-1.718 -1.1923 TD
[(uorescent)-398.9(prot)-6.5(ein)-397.9(\(GFP\))-395.2(transf)-8.1(ected)-398.1(cells)-397.3(were)-401.7(killed.)]TJ
0 -1.1869 TD
[(CRISPR-Cas9)-455.7(transfected)-454.9(cells)-451.5(were)-450.5(then)-445.2(dilute)-7.9(d)-443(t)0(o)-449.2(a)]TJ
0 -1.1923 TD
[(concentratio)-11.2(n)-296.7(o)0(f)-305.1(1)-296.7(cell)-305.4(per)-302.5(well)-305.3(and)-298.1(the)-305.7(resulta)-8.7(nt)-297.5(single-)]TJ
T*
[(cell)-294.5(clones)-296.1(amplied,)-296.6(replica)-299(plated,)-296.4(and)-292.7(DNA)-290.2(extra)-8.1(cted)]TJ
0 -1.1869 TD
[(for)-302.7(screening.)]TJ
/F9 1 Tf
/Cs8 cs 1 scn
0 -2.6556 TD
[(Cas9)-298.5(ribonuclear)-299.9(protein)-299.6(delivery)]TJ
/F7 1 Tf
0 0 0 rg
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[(SgRNAs)-330.6(were)-331.2(synthesized)-334.7(using)-327(an)-332(Engen)-333.1(sgRNA)-329.8(syn-)]TJ
0 -1.1923 TD
[(thesis)-251.9(kit)-255(\(New)-260.7(England)-251.2(Biolabs\))-254.4(according)-256(to)-254.1(the)-257(manu-)]TJ
0 -1.1869 TD
[(facturers)-198.6(instructions)-198.2(and)-200.6(puried)-198.3(using)-202.4(Zymo-Spin)]TJ
/F10 1 Tf
20.7896 0 TD
()Tj
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1.0893 0 TD
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(IC)Tj
-21.879 -1.1923 TD
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[(C)21.1(o)23.1(l)15.4(u)23.1(m)22.3(n)17.7(s)-319.6(\(Zymo)-339.5(Resea)7.4(rch;)-341.3(see)-341.4(Supple)10.6(mentary)-336.7(Table)-334.1(S1)]TJ
T*
[(for)-232.3(sequence)9.4(s\).)-235.1(A)-241.3(non-t)11.2(argeting)-233.7(control)-225.5(sg-RNA)-235.2(supplie)10.8(d)]TJ
0 -1.1869 TD
[(with)-230.7(the)-235.3(Engen)-224.7(kit)-233.3(uses)-234(a)-235.9(sequen)9.6(ce)-238.7(from)-231.7(the)-229.9(tetR\(C\))-228.5(gene)]TJ
0 -1.1923 TD
[(in)-384.2(the)-381.6(pBR322)-380.4(plasm)9.4(id)-384.2(that)-382.4(is)-385.4(not)-382.8(found)-382.2(in)-384.2(the)-381.6(human)]TJ
/F6 1 Tf
9.9626 0 0 9.9626 59.7543 753.8455 Tm
[(CR)-7.6(ISPR-Ca)-10.9(s9)-301.4(CAUSES)-304.6(CHR)-9.2(OMOSOM)-12.2(AL)-301.8(INSTAB)-11.1(ILITY)-24687.5(407)]TJ
ET
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COIKPE+AdvPSMP10 qJ
AdvPSMP10 M iJQ17pjjfjzu:aӕzkzbntDTN~?$Mis
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/GS1 gs
BT
/F7 1 Tf
10.4607 0 0 10.4607 62.022 698.2297 Tm
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[(genome.)-200.7(The)-205.8(most)-199.1(similar)-202.6(human)-204(sequen)9.6(ce)-206.2(has)-207.2(three)-203.2(mis-)]TJ
0 -1.1923 TD
[(matches.)-331.6(RNP)-329.4(complex)9.2(es)-338.6(with)-333.6(Cas9)-332.4(protein)-328.4(\(New)-331.2(Eng-)]TJ
T*
[(land)-385.6(Biolabs)8.8(\))-387.8(and)-384.8(synthesi)11(zed)-389(sgRNAs)-383.8(were)-385.4(made)-383(and)]TJ
0 -1.1869 TD
[(mixed)-222.5(with)-219.8(HDR)-223(templat)10.1(e)-225(\(Suppleme)10.8(ntary)-220.6(Table)-220.3(S2\))-224.6(im-)]TJ
0 -1.1923 TD
[(mediate)8.1(ly)-438.4(prior)-426.8(to)-438.4(transfe)10.8(ction)-435.2(by)-430.8(electropo)7.3(ration)-432.8(with)]TJ
T*
[(an)-592.2(Amaxa)-586.4(4D-Nucleof)7.8(ector)-588(\(Lonza\),)-586.5(carried)-588.4(out)-588.8(us-)]TJ
0 -1.1869 TD
.0156 Tc
[(i)15.6(n)15.6(g)-524.4(pro)6.2(g).7(ram)-542(C)-1.3(M-150)-546.6(and)-541.2(S)-1.5(G)-545.4(cell)-543.4(lin)6.2(e)-545.4(k).7(it)-543.4(\(L)-4.5(o)6.2(n).7(za\))]TJ
0 -1.1923 TD
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[(according)-541.5(t)-1.8(o)-541.5(t)3.6(he)-545.7(manufacturers)-542.7(instru)5.9(ctions.)-542.2(F)3.7(orty-)]TJ
T*
.0149 Tc
[(eight)-435.7(hours)-440.1(post)-435.7(t)-2.2(ransfection,)-434.2(cells)-440.1(w)1.3(ere)-437.8(d)0(iluted)-439(to)-439(a)]TJ
0 -1.1869 TD
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[(conc)-4.6(entr)-4.8(ation)-228(o)5.1(f)-227(1)-228(cell)-224.8(p)-.4(er)-227(well)-224.8(and)-228(t)2.8(he)-226.8(re)-4.6(sultant)-224.8(single-)]TJ
0 -1.1923 TD
0 Tc
[(cell)-294.5(clones)-296.1(amplied,)-296.6(replic)-8.9(a)-290.1(plated,)-296.4(and)-292.7(DNA)-290.2(ex)-6.8(tracted)]TJ
T*
[(for)-302.7(screenin)-9.3(g.)]TJ
/F9 1 Tf
/Cs8 cs 1 scn
0 -2.3684 TD
[(Mutation)-298.3(screening)-302.9(by)-297.8(PCR)-300.8(and)-298.4(Sanger)-298.1(sequencing)]TJ
/F7 1 Tf
0 0 0 rg
0 -1.2032 TD
[(PCR)-256.7(wa)10.6(s)-265.4(u)0(s)12.4(e)0(d)-261.6(t)0(o)-259.6(amp)15.4(lif)11.6(y)-264.2(t)0(h)11.4(e)-268.4(ta)12.6(rge)11.8(t)-266.4(l)0(o)11.4(c)0(i)-258.4(\(se)16.1(e)-268.4(S)0(u)11.4(ppl)12.8(em)14(en-)]TJ
0 -1.1869 TD
[(tar)15(y)-361.7(T)0(a)9.6(b)0(l)11.4(e)-360.5(S1)-357.1(for)-351.5(pri)13.8(me)14(rs\))10.4(.)-357(P)0(u)11.4(ri)11.6(ed)-353.7(PCR)-348.9(pro)16(duc)10.8(ts)-352.9(we)10.6(re)]TJ
0 -1.1923 TD
[(seq)15(uen)16.2(ce)10.6(d)-708.6(b)0(y)-696.3(s)0(t)10.2(and)16.2(ar)10.4(d)-703.2(San)14(ger)-692.7(tec)15.2(hno)15(lo)11.4(gy)-701.8(\(Z)14.8(ool)12.8(og)13.6(y)]TJ
T*
[(Seq)14(uen)16.2(ci)12.6(ng)-506.6(fa)10.4(cil)17.2(ity)10.6(,)-508.7(U)0(n)14.9(ive)14(rsi)12.6(ty)-503.4(of)-500.2(Oxf)11.8(or)14.6(d\).)-499.5(Seq)14(uen)16.2(ce)10.6(s)]TJ
0 -1.1869 TD
[(we)10.6(re)-211.8(ana)17.4(ly)11.4(zed)-204.8(for)-205.2(qu)13.6(ali)11.8(ty)-205.4(of)-207.6(tr)12.4(ace)13.2(,)-216.1(a)0(l)12.6(i)0(g)11.4(ned)-206(to)-205.4(re)10.4(fer)12.8(enc)12(e)-208.8(se-)]TJ
0 -1.1923 TD
[(que)16.2(nce)12(s)-303.3(wit)11.8(h)-302.1(the)-300.3(op)13.6(en)-299.5(sou)13.8(rc)10.4(e)-306.3()51.2(A)-298.5(pla)14(sm)11.6(id)-302.9(Edi)13(to)11.4(r)59()-306.5(\()0(A)10.4(p)0(E)]TJ
T*
[(v2.)14.3(0.)12.9(55\))16(,)-389.5(and)-379.4(ma)14(nua)16.2(lly)-379.6(in)11.4(spe)15(cte)15.2(d)-388.8(for)-378.6(des)15(ire)15(d)-388.8(and)-379.4(un)13.6(de-)]TJ
0 -1.1869 TD
[(si)10.2(re)10.4(d)-296.7(seq)15(uen)16.2(ce)-292.9(cha)17.4(nge)16.2(s)-297.9(b)0(y)-289.9(t)0(w)12.6(o)-296.7(in)11.4(dep)16.2(end)16.2(ent)-284.1(re)10.4(sea)10.8(rc)10.4(her)17.2(s.)]TJ
/F9 1 Tf
/Cs8 cs 1 scn
0 -2.3684 TD
[(Preparation)-217.4(of)-214(metaphase)-212.2(spreads)-215.6(and)-211.7(DAPI)-212.9(staining)]TJ
/F7 1 Tf
0 0 0 rg
0 -1.2032 TD
[(Ch)11.6(rom)15.2(os)12.4(ome)-212.2(sp)12.4(rea)13(ds)-215.2(we)10.6(re)-217.2(har)11.8(ves)15(te)12.6(d)-220.8(u)0(s)12.4(ing)-214.8(st)10.2(an)14.8(dar)11.8(d)-220.8(t)0(e)12.6(ch-)]TJ
0 -1.1923 TD
[(ni)11.4(que)16.2(s.)-264.7(Br)12.6(ie)11.8(y,)-258(Co)11.6(lce)15.2(mi)10.6(d)-264.2(\(Th)10.8(er)10.4(mo)-258.2(Fis)14.9(her)-253.7(Sci)11.8(ent)14(i)14.6(c\))-260.6(was)]TJ
0 -1.1869 TD
[(add)16.2(ed)-288.7(to)-286.7(sub)13.8(con)16.2(u)11.4(ent)-284.1(cul)14(tu)11.4(re)10.4(s)-292.5(a)0(t)-290.9(a)-290.1(n)11.4(al)-290.9(con)16.2(ce)10.6(nt)11.4(rat)9.6(io)11.4(n)-291.3(o)0(f)]TJ
0 -1.1923 TD
[(50)-154.4(ng/)12.8(mL)-192.9(for)-188.9(3)-161.2(h)0(.)-193(M)0(e)9.6(t)0(a)12.6(pha)10.8(se)13.6(s)-200.3(w)0(e)10.6(r)0(e)-195.5(det)14(ac)10.6(hed)-189.7(by)-192.3(mi)10.6(ld)-194.5(try)13.8(p-)]TJ
T*
[(si)10.2(ni)11.4(zat)15.2(ion)-193.1(and)-195.1(swo)15.1(lle)17.2(n)-204.6(i)0(n)-199.9(h)0(y)13.6(pot)18.2(oni)12.8(c)-203.4(s)0(o)12.4(l)0(u)11.4(tio)10.6(n)-199.1(\()0(K)10.4(C)0(l)-202(7)0(5)-154.4(m)0(M)13(\))]TJ
0 -1.1869 TD
[(bef)17.2(ore)-232.1(bei)14(ng)-235.7(x)11.4(ed)-239.9(tw)12.6(ice)-234.1(in)-237.9(Ca)12.8(rno)16(ys)-234.4(x)11.4(at)12.6(iv)11.4(e.)-240.6(Tw)9.6(en)14.8(ty)-237.9(mi)10.6(-)]TJ
0 -1.1923 TD
[(cr)10.4(ol)11.4(ite)11.8(rs)-387.6(of)-381(the)-381.6(cel)9.8(l)-391(s)0(u)12.4(s)0(p)12.4(ens)15(io)11.4(n)-394.2(w)0(a)10.6(s)-390(dro)16(ppe)16.2(d)-394.2(o)0(n)13.6(t)0(o)-384.2(cle)9.8(an)]TJ
T*
[(sl)10.2(id)11.4(es)-517.5(an)14.8(d)-518.9(all)11.8(owe)17.5(d)-518.9(t)0(o)-514.3(d)0(r)14.6(y)-518.9(ove)16.2(rni)13.8(gh)13.6(t.)-515(The)-509.3(sl)10.2(id)11.4(es)-512.1(we)10.6(re)]TJ
0 -1.1869 TD
[(mo)12.8(unt)12.8(ed)-673.5(in)-676.9(DAP)15.3(I/V)15(ect)9.8(as)13.6(hi)11.4(eld)-674.3(\(V)10.4(ec)10.6(tor)-674.5(La)9.6(bo)13.6(ra)10.4(tor)13.8(ies)12.8(\).)]TJ
0 -1.1923 TD
[(Ima)16.4(ges)-391.4(we)10.6(re)-390.6(col)14(lec)15.2(ted)-387(wit)11.8(h)-394.2(a)0(n)-391.6(Oly)14(mp)12.8(us)-394(BX)12.8(-51)-390.4(mi)10.6(cr)10.4(o-)]TJ
T*
[(sco)15(pe,)-298.8(equ)16.2(ip)11.4(ped)-298.1(wit)11.8(h)-307.5(a)-306.3(JAI)-298.3(CVM)14.5(4)]TJ
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[(CC)9.6(D)-311.7(c)0(a)10.6(mer)16.4(a,)-305.6(us)12.4(in)11.4(g)]TJ
-14.931 -1.1869 TD
[(Le)9.6(ica)-304.5(Cy)11.6(to)11.4(vi)11.4(si)10.2(on)-306.1(Ge)10.6(nus)-300.5(v7)13.6(.1.)-300.7(Chr)14(om)12.8(oso)13.8(me)-305.7(nu)13.6(mbe)15.4(r)-311.9(w)0(a)10.6(s)]TJ
0 -1.1923 TD
[(ana)12(ly)11.4(ze)10.6(d)-296.7(i)0(n)-286.7(a)-295.5(mi)10.6(ni)11.4(mum)-284.7(of)-288.9(25)-289.9(met)13.2(aph)16.2(ase)16.2(s)-297.9(per)-280.8(cel)9.8(l)-293.5(lin)16(e.)]TJ
/F9 1 Tf
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(FISH)Tj
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[(BAC)-340(probes)-337.4(that)-344.5(were)-336.6(not)-339.5(available)-341.5(in-house)-336.9(were)-342.1(pur-)]TJ
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[(chased)-282(from)-280.5(SourceBioscience.)-281.2(The)-281.8(plasmids)-277.2(containing)]TJ
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0 Tc
[(chromosome-specic)-295(centromeric)-297.4(DNA)-295.7(were)-293.3(a)-295.5(kind)-294.7(gift)]TJ
0 -1.1923 TD
[(from)-253.3(Prof.)-254(Mariano)-248.9(Rocchi)-253.6(\(Bari)-254.2(University,)-255.3(Bari,)-250.4(Italy\).)]TJ
T*
[(The)-373.8(probes)-370(were)-374.6(labeled)-370.4(with)-377(the)-370.8(Nick)-373.6(Translation)-368.2(Kit)]TJ
0 -1.1869 TD
[(\(Abbott)-355.5(Molecular\))-358.6(according)-359(to)-357.1(the)-354.5(manufacturers)-360.6(in-)]TJ
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[(structions,)-349.8(with)-355.3(biotinylated-16)7.9(-dUTP)-355.9(\(SigmaAldrich\))7.1(,)]TJ
T*
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[(Spect)-4.7(r)4(um)-225.5(Red-dUTP)-232.3(\(Vysis,)-225.3(A)-1.2(bbott)-232.3(M)3.2(olecular\),)-225.3(Spectrum-)]TJ
0 -1.1869 TD
0 Tc
[(Green)-400.3(dUTP)-396.2(\(Vysis,)-399.7(Abbott)-401.3(Molecul)-8.3(ar\),)-398.4(and)-395.7(Spectrum)]TJ
0 -1.1923 TD
[(Gold-dUT)-9.1(P)-320.6(\(Enzo)-326.6(Laborat)-10.5(ories\).)-327.1(The)-325(probe)-7.1(s)-319.6(were)-331.2(puri-)]TJ
T*
[(ed)-370.8(by)-371.2(preci)-8.1(pitation,)-375.1(adding)-372(a)-371.4(1)0(0)]TJ
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()Tj
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[(excess)-375.2(of)-370.2(unlabe)-7.7(led)]TJ
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[(Cot1)-275(DNA)-268.5(\(Thermo)-274.9(Fis)-6.8(her)-270(Scienti)-8.3(c\),)-270.7(and)-271(re-suspend)-11.1(ed)]TJ
0 -1.1923 TD
[(in)-433(hybridiz)-7.3(ation)-435.2(buffer)-437.2(\(50%)-430(formamide,)-441.4(10%)-432.4(dextran)]TJ
23.998 60.7159 TD
[(sulf)-7.6(ate,)-526(2)]TJ
/F10 1 Tf
4.1243 0 TD
()Tj
/F7 1 Tf
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[(SSC\).)-524.8(The)-525.5(labele)-8.7(d)-518.9(probe)-7.1(s)-520.1(were)-526.3(denatured)]TJ
-4.8397 -1.1923 TD
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[(for)-358.8(8)-164.8(min)-359.9(a)-1(t)-356.7(8)3.3(5)]TJ
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()Tj
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[(C)-356.6(i).8(n)-360.1(a)-358.9(ther)4.1(mocycler)-359.1(machine.)-360.8(The)-358.9(B)1(AC/)]TJ
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[(PAC)-302.3(probe)-7.1(s)-297.9(were)-309.5(pre-anneale)-11.3(d)-296.7(a)0(t)-301.7(3)0(7)]TJ
/F10 1 Tf
14.9852 0 TD
()Tj
/F7 1 Tf
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[(C)-304.1(for)-297.3(30)-170.6(min.)]TJ
-14.3295 -1.1869 TD
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[(Meta)-5.4(pha)-5.4(s)3.1(e)-704.5(s)-2.3(pread)-705.7(DNA)-704.5(was)-706.9(d)-1.1(enature)-5.4(d)-700.3(in)-700.3(Na)-5.4(OH,)]TJ
-1.0514 -1.1923 TD
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[(0.07)-162.6(M,)-282.6(fo)5.4(r)-286.3(2)-162.6(min)5.4(.)-282.6(Following)-281.9(d)0(ehydration)-281.9(i)-2.3(n)-281.9(a)1.2(n)-281.9(a)-4.3(lcohol)-284.1(se-)]TJ
T*
.4431 Tc
[(r)456.3(i)464(e)462(s)459.6(,t)458.5(h)466.2(e)-4.1(d)460.8(e)461.9(n)460.8(a)461.9(t)458.5(u)466.2(r)461.8(e)456.5(dp)460.8(r)461.8(o)460.8(b)466.2(e)-4.1(m)459.9(i)464(xw)462(a)461.9(s)-1.1(a)456.5(p)466.2(p)460.8(l)458.5(i)464(e)456.5(dt)464(ot)458.5(h)460.8(e)1.3(s)459.6(l)464(i)458.5(d)460.8(e)462(s)]TJ
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[(under)-308.2(a)-308(cov)5.2(e)-4.5(rslip)5.2(.)-309.9(Th)5.2(e)-308(hybridization)-303.7(w)1(as)-304.9(carried)-309.2(o)5.2(ut)-306(over-)]TJ
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[(night)-332.9(at)-332.9(37)]TJ
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()Tj
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[(C.)-337(Post-hybrid)5.2(ization)-336.3(washes)-337.5(w)1(ere)-335.1(c).9(arried)-330.8(out)]TJ
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[(in)-209.1(0)7.7(.)1.6(1)]TJ
/F10 1 Tf
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()Tj
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[(SS)-5.4(C)-211.2(a)-2.1(t)-216.8(6)-3.2(0)]TJ
/F10 1 Tf
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0 Tc
()Tj
/F7 1 Tf
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T*
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T*
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T*
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[(Cas9)-348.7(machinery)-343.5(to)-346.3(drive)-350.7(large-scale)-350.8(chromosomal)-343.9(rear-)]TJ
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T*
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[(available)-525.8(CRISPR-Cas9)-526.1(mutated)-524.2(clone)-523.9(of)-516.5(the)-522.5(diploid)]TJ
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[(CIN)-272(cell)-278.3(line)-274(Hct11)-9.1(6.)-268.9(Simil)-8.1(ar)-271.4(to)-275.8(HCC2998,)-274.1(no)-273.6(large)-279.1(dif-)]TJ
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[(ferences)-622.7(in)-611.8(chrom)-7.9(osome)-617.1(number)-613.5(or)-619.5(karyotype)-620.3(were)]TJ
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[(FI)-7(G.)-330.6(1.)]TJ
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[(CRISP)-9.6(R-Cas)-8.6(9)-298.1(mutati)-11.1(on)]TJ
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