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T*
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T*
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T*
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T*
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T*
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[(\(pNaive,)-431.5(pTargeted,)-428.5(or)-429.8(pPriming\),)-431.1(then)-429(plated)-427.2(on)-430.8(agar)]TJ
T*
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()Tj
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T*
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(C)Tj
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[(\(shaking\),)-247(and)-238.5(then,)-244(the)]TJ
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(cas)Tj
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(L)Tj
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[(-arabinose)-558.3(and)-558.2(0.5)-164.5(mM)-556(of)]TJ
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[(WU)-304.5(ET)-299.4(AL.)]TJ
ET
endstream
endobj
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<>stream
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[(corrected)-535.6(culture)-527.8(were)-531.7(harvested)-533.6(by)-528.3(centrifugation)-531(at)]TJ
T*
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[(reactions)-557.5(using)-560(the)-555(Q5)-559.6(High-Fidelity)-555.3(2)]TJ
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[(\(New)-423.3(England)-419.2(Biolabs\))-422.4(and)-422.8(with)-420.3(forward)-418.3(primers)-419.4(that)]TJ
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()Tj
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[(nucleotide)-303.4(mismatch)]TJ
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[(with)-279.4(the)-273.2(rst)-275.4(base)-275(of)-272.6(the)-278.6(existing)-271.8(spacer.)-274.7(This)-274(PCR)-278.4(strat-)]TJ
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[(egy)-357.7(allows)-357.9(preferential)-356(amplication)-354.6(of)-353.9(expanded)-359.2(com-)]TJ
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[(pared)-369(with)-366.2(unexpanded)-367.2(arrays)-367.8(but)-366.6(with)-366.2(the)-365.4(trade-off)-368(of)]TJ
T*
[(not)-214.8(detecting)-212.7(new)-210.2(spacers)-215.6(beginning)-207.4(with)-214.4(the)-213.6(same)-210.8(nucle-)]TJ
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[(otide)-299.7(as)-300.7(the)-300.3(existing)-298.9(spacer.)]TJ
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(26,27)Tj
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10.7525 0 TD
()Tj
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[(degenerate)-225.1(base)-226.2(by)-219.4(exonu-)]TJ
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[(clease)-272(activity)-266.3(of)-267.2(the)-267.8(Q5)-267(DNA)-268.5(polymerase,)-266.7(which)-263.8(would)]TJ
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[(result)-201(in)-199.9(loss)-199.5(of)-202.1(primer)-202.8(specicity)-198.4(for)-199.7(expanded)-202(arrays,)-199(we)]TJ
0 -1.1923 TD
[(included)-283.5(a)-279.2(phosphorothioate)-274.7(bond)-281.6(before)-279.9(the)-278.6(3)]TJ
/F10 1 Tf
18.8223 0 TD
()Tj
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(degener-)Tj
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[(ate)-353.3(base;)-346.3(we)-347.1(observed)-349.7(that)-344.5(PCRs)-348.6(using)-348.7(primers)-343.5(without)]TJ
0 -1.1869 TD
[(this)-579.9(phosphorothioate)-572.8(modication)-577.2(were)-575.1(less)-577.7(specic)]TJ
0 -1.1923 TD
[(for)-253.9(expanded)-256.2(versus)-251.9(no)13.6(nex)16.2(pan)10.8(de)14.8(d)-253.3(a)0(r)10.4(r)0(a)10.4(y)0(s)-242.3(\()0(S)12.4(upp)15(lem)13.2(ent)14(ar)10.4(y)]TJ
0 -1.1869 TD
[(Fig)16(.)-362.4(S)0(2)11.4(\))0(.)-354.6(P)0(C)14.9(R)0(s)-358.1(a)0(n)14.8(d)-361.7(th)11.4(er)10.4(mocycling)-364.2(conditions)-361.2(were)-363.7(car-)]TJ
0 -1.1923 TD
[(ried)-287.1(out)-285.3(according)-283.1(to)-281.2(the)-289.5(manufacturers)-279.3(protocol.)-283(Initial)]TJ
T*
[(denaturation)-207.5(at)-204.2(95)]TJ
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7.1593 0 TD
()Tj
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[(C)-206.6(was)-205.9(10)-165.2(min,)-204.1(extension)-202(time)-209.8(30)-165.2(s,)-205(and)]TJ
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[(the)-359.9(annealing)-362.2(temperature)-357(was)-363.1(67)]TJ
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[(C)-358.3(and)-357.7(70)]TJ
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3.8317 0 TD
()Tj
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[(C)-358.3(for)-356.9(V-A)]TJ
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[(and)-536.6(V-B,)-536.8(respectively.)-537.3(Amplicons)-533.4(were)-537.2(separated)-537.8(by)]TJ
T*
[(gel)-300.3(electrophoresis)-301(using)-299.9(3%)-298.3(agarose)-301.1(gels.)]TJ
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[(Illumina)-299.3(MiSeq)-301.2(sequencing)-296.9(of)-300.7(spacer)-297.4(acquisition)]TJ
/F7 1 Tf
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[(To)-311.4(prepare)-309.8(the)-311.2(samples)-307.6(for)-308.1(high-throughput)-303.4(sequencing,)]TJ
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[(PCR)-462.7(products)-456.1(were)-461.3(puried)-463.9(by)-457.9(spin-column)-454.9(clean-up,)]TJ
0 -1.1923 TD
[(and)-406.5(then)-407.3(quantied)-398.1(using)-408.3(a)-403.9(Qubit)-402.7(dsDNA)-407.8(broad)-402.7(range)]TJ
T*
[(assay.)-230.7(Equal)-232.3(molar)-232.3(amounts)-226.3(of)-229.2(product)-234.1(from)-226.3(each)-234.7(sample)]TJ
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[(were)-249.9(pooled)-247.3(into)-248.1(two)-251.5(libraries,)-250.3(keeping)-244.7(the)-251.5(type)-250.1(V-A)-247.1(and)]TJ
0 -1.1923 TD
[(V-B)-477.9(samples)-475.6(separate.)-478.9(Sequencing)-474.6(libraries)-473.2(were)-477.6(pre-)]TJ
T*
[(pared)-439.4(with)-442(the)-441.2(NEB)-440.8(Next)-438.6(Ultra)-438.4(II)-439.6(DNA)-442(Library)-438.8(Prep)]TJ
0 -1.1869 TD
[(Kit)-335(and)-330.6(the)-332.8(NEBNext)-332(Multiplex)-330.9(Oligo)-332.2(Index)-332.2(primer)-327.4(set)]TJ
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[(1,)-480.2(according)-478.2(to)-476.3(the)-473.7(manufacturers)-479.8(protocol.)-478.1(Libraries)]TJ
T*
[(were)-212(pooled)-209.4(equally)-214.4(and)-206(spiked)-210.6(with)]TJ
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14.7359 0 TD
(*)Tj
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[(20%)-210.2(of)-207.6(PhiX)-214.4(con-)]TJ
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[(trol)-295.9(library)-294.9(\(Illumina\),)-292(then)-288.1(sequenced)-298.1(using)-289.1(an)-294.1(Illumina)]TJ
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[(MiSeq)-516.3(v3)-512.1(ow)-511.6(cell)-516.7(\(2)]TJ
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[(300)-516.1(base)-513.5(paired-end\).)-510.6(Image)]TJ
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[(analysis,)-399(base)-394.2(calling,)-394.8(demultiplexing,)-395.7(and)-395.7(data)-395.3(quality)]TJ
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[(Contaminant)-425.2(reads)-435.2(from)-432.2(PhiX,)-425(the)-435.8(plasmids)-428.8(present)]TJ
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[(in)-470.9(the)-468.3(assay)-469.9(\(with)-466.7(the)-468.3(mini-CRISPR)-469.7(loci)-469.1(masked\),)-465(or)]TJ
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(the)Tj
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1.5663 0 TD
[(E.)-333.7(coli)]TJ
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3.0404 0 TD
[(genomes)-331.2(were)-336.6(removed)-334.5(by)-333.2(mapping)-333.5(the)-332.8(raw)]TJ
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[(fastq)-379(reads)-381(to)-378.8(the)-376.2(respective)-381.5(elements)-376.4(with)-377(BowTie2.)]TJ
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(28)Tj
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[(The)-617.7(remaining)-617.9(paired-end)-616.7(reads)-619.5(were)-618.5(merged)-617.7(using)]TJ
T*
[(paired-end)-329.5(read)-332.4(merger)-329.4(\(PEAR\).)]TJ
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(29)Tj
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[(c)13.4(l)0(u)16.8(d)12.2(ed)-538(in)-536(th)22.3(e)-550.2(p)12.2(r)13.2(i)0(m)16(e)13.4(rs)-539.3(u)12.2(s)11(ed)-538(to)-536(g)12.2(e)0(n)20.3(e)0(r)15.8(a)13.4(te)-540.2(th)22.3(e)-550.2(e)13.4(xp)19.1(an)20.3(d)12.2(e)0(d)]TJ
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T*
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T*
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(31)Tj
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T*
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T*
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T*
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T*
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T*
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ET
endstream
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62.022 705.316 m
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[(electroph)-14(oresis.)]TJ
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[(genes)-306.4(\(and)-305.5(variation)-9.9(s)-299.2(thereof)-90.4(\).)-301.6(CRISP)-9.6(R-)]TJ
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[(muta)-10.1(ted)-300.4(Cas4.)]TJ
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(Cas4)Tj
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