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Yuemei Dong, Maria L. Simões, Eric Marois, George Dimopoulos
CRISPR/Cas9 -mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection
www.plospathogens.org
www.plospathogens.org
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[(\n)30()6()11()10(\r)6()15()4()8()]TJ
/F8 1 Tf
-32.6208 -1.2983 Td
(with)Tj
/F9 1 Tf
1.9955 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
0.9921 0 Td
[( )9()11()4(!)1()10()4()16()-9( )12()]TJ
/F8 1 Tf
(,)Tj
/F9 1 Tf
4.7055 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
0.9921 0 Td
[()-13()15()3()]TJ
/F8 1 Tf
(,)Tj
2.5342 0 Td
(and)Tj
/F9 1 Tf
1.7291 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
0.9921 0 Td
[()4()2()6()-2(\r)6()2()]TJ
/F8 1 Tf
(,)Tj
3.2542 0 Td
(suggesting)Tj
4.405 0 Td
(that)Tj
1.7745 0 Td
(FREP1)Tj
2.9764 0 Td
(might)Tj
2.5965 0 Td
(be)Tj
1.1508 0 Td
(a)Tj
0.652 0 Td
(promising)Tj
4.3426 0 Td
(trans-)Tj
-35.0926 -1.2983 Td
(mission-blocking)Tj
7.1602 0 Td
(vaccine)Tj
3.1975 0 Td
(\(TBV\))Tj
2.7836 0 Td
(target)Tj
2.5115 0 Td
([)Tj
0.83 0.64 0.02 0 k
(23)Tj
0 g
(].)Tj
2.0749 0 Td
(While)Tj
2.6532 0 Td
(such)Tj
2.058 0 Td
(a)Tj
0.6519 0 Td
(TBV)Tj
2.109 0 Td
(has)Tj
1.542 0 Td
(already)Tj
3.1125 0 Td
(been)Tj
2.1203 0 Td
(developed,)Tj
-31.9746 -1.3039 Td
(here)Tj
1.9672 0 Td
(we)Tj
1.3266 0 Td
(have)Tj
2.0579 0 Td
(taken)Tj
2.4208 0 Td
(a)Tj
0.652 0 Td
(different)Tj
3.6566 0 Td
(interference)Tj
5.0343 0 Td
(approach,)Tj
4.1783 0 Td
(using)Tj
/F9 1 Tf
2.3867 0 Td
[()]TJ
/F8 1 Tf
2.8914 0 Td
(gene)Tj
2.0806 0 Td
(disruption)Tj
4.4106 0 Td
(to)Tj
1.0262 0 Td
(investi-)Tj
-34.0892 -1.2982 Td
(gate)Tj
1.8425 0 Td
(its)Tj
1.1451 0 Td
(biological)Tj
4.0876 0 Td
(role)Tj
1.7688 0 Td
(as)Tj
1.0148 0 Td
(a)Tj
0.6519 0 Td
(host)Tj
1.9163 0 Td
(factor)Tj
2.5398 0 Td
(and)Tj
1.7348 0 Td
(its)Tj
1.1452 0 Td
(potential)Tj
3.7587 0 Td
(as)Tj
1.0148 0 Td
(a)Tj
0.6519 0 Td
(transmission-blocking)Tj
9.1786 0 Td
(target)Tj
2.5114 0 Td
(for)Tj
-34.9622 -1.2983 Td
(the)Tj
1.4683 0 Td
(development)Tj
5.4085 0 Td
(of)Tj
1.0148 0 Td
(a)Tj
0.6576 0 Td
(novel)Tj
2.3981 0 Td
(malaria)Tj
3.2258 0 Td
(control)Tj
3.1237 0 Td
(strategy.)Tj
-16.1006 -1.3039 Td
(To)Tj
1.3379 0 Td
(generate)Tj
3.6113 0 Td
(CRISPR/Cas9-based)Tj
/F9 1 Tf
8.3678 0 Td
[()]TJ
/F8 1 Tf
2.8914 0 Td
(knockout)Tj
/F9 1 Tf
4.0251 0 Td
[(\n)]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
1.0942 0 Td
[()2()16()17()11()4()6()]TJ
/F8 1 Tf
3.5206 0 Td
(mosquitoes,)Tj
5.023 0 Td
(we)Tj
1.3266 0 Td
(established)Tj
4.5977 0 Td
(a)Tj
-36.9918 -1.2983 Td
(modified)Tj
3.8437 0 Td
(CRISPR/Cas9)Tj
5.7656 0 Td
(gene)Tj
2.0807 0 Td
(editing)Tj
3.0217 0 Td
(method)Tj
3.3222 0 Td
(based)Tj
2.4717 0 Td
(on)Tj
1.2756 0 Td
(crossing)Tj
3.5263 0 Td
(a)Tj
0.652 0 Td
(guide)Tj
2.4321 0 Td
(RNA)Tj
2.262 0 Td
(\(gRNA\)-express-)Tj
-30.6536 -1.2983 Td
(ing)Tj
1.5023 0 Td
(transgenic)Tj
4.3597 0 Td
(line)Tj
1.7064 0 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.6519 0 Td
(transgenic)Tj
4.3597 0 Td
(line)Tj
1.7065 0 Td
(expressing)Tj
4.4276 0 Td
(Cas9)Tj
2.1487 0 Td
(under)Tj
2.6192 0 Td
(the)Tj
1.474 0 Td
(control)Tj
3.1181 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.474 0 Td
(germline-)Tj
-32.5585 -1.3039 Td
(specific)Tj
/F9 1 Tf
3.1917 0 Td
[(")92()-3(#)]TJ
/F8 1 Tf
2.6476 0 Td
(promoter)Tj
4.0365 0 Td
([)Tj
0.83 0.64 0.02 0 k
(24)Tj
0 g
(].)Tj
2.0749 0 Td
(Mosquitoes)Tj
4.8756 0 Td
(carrying)Tj
3.5546 0 Td
(the)Tj
1.4684 0 Td
(desired)Tj
3.1237 0 Td
(knockout)Tj
4.0252 0 Td
(mutation)Tj
3.9174 0 Td
(were)Tj
2.126 0 Td
(iso-)Tj
-35.0416 -1.2982 Td
(lated)Tj
2.1486 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4683 0 Td
(progeny.)Tj
3.7304 0 Td
(The)Tj
1.7858 0 Td
(resulting)Tj
/F9 1 Tf
3.7304 0 Td
[()]TJ
/F8 1 Tf
2.8856 0 Td
(knockout)Tj
4.0252 0 Td
(mutant)Tj
3.1464 0 Td
(mosquitoes)Tj
4.8019 0 Td
(showed)Tj
3.2485 0 Td
(a)Tj
0.6576 0 Td
(significant)Tj
-32.6662 -1.2983 Td
(decrease)Tj
3.6056 0 Td
(in)Tj
1.0375 0 Td
(their)Tj
2.1089 0 Td
(permissiveness)Tj
6.1795 0 Td
(to)Tj
/F9 1 Tf
1.0262 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
0.9921 0 Td
[( )9()11()4(!)1()10()4()16()-9( )12()]TJ
/F8 1 Tf
4.473 0 Td
(and)Tj
/F9 1 Tf
1.7348 0 Td
[()]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
0.9921 0 Td
[()4()2()6()-2(\r)6()2()]TJ
/F8 1 Tf
3.0274 0 Td
(infection.)Tj
4.0195 0 Td
(However,)Tj
4.0762 0 Td
(the)Tj
-33.2728 -1.3039 Td
(mutants)Tj
3.5035 0 Td
(also)Tj
1.7689 0 Td
(exhibited)Tj
3.9288 0 Td
(a)Tj
0.6519 0 Td
(significant)Tj
4.388 0 Td
(fitness)Tj
2.7723 0 Td
(cost)Tj
1.8085 0 Td
(in)Tj
1.0318 0 Td
(terms)Tj
2.4945 0 Td
(of)Tj
1.0148 0 Td
(blood-feeding)Tj
5.8109 0 Td
(propensity,)Tj
4.7112 0 Td
(longevity)Tj
-33.8851 -1.2983 Td
(after)Tj
2.0352 0 Td
(blood)Tj
2.5115 0 Td
(meal,)Tj
2.3697 0 Td
(fecundity,)Tj
4.2066 0 Td
(and)Tj
1.7292 0 Td
(egg)Tj
1.576 0 Td
(hatching)Tj
3.7077 0 Td
(rates,)Tj
2.3357 0 Td
(but)Tj
1.5534 0 Td
(no)Tj
1.2756 0 Td
(impact)Tj
2.982 0 Td
(on)Tj
1.2756 0 Td
(the)Tj
1.4683 0 Td
(adult)Tj
2.2507 0 Td
(mosquito)Tj
4.0139 0 Td
(size)Tj
-35.2911 -1.2982 Td
(or)Tj
1.0941 0 Td
(on)Tj
1.2756 0 Td
(the)Tj
1.474 0 Td
(life-span)Tj
3.6567 0 Td
(of)Tj
1.0148 0 Td
(mosquitoes)Tj
4.7962 0 Td
(maintained)Tj
4.7905 0 Td
(on)Tj
1.2756 0 Td
(a)Tj
0.6519 0 Td
(10%)Tj
1.9276 0 Td
(sugar)Tj
2.3811 0 Td
(solution.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 469.0204 cm
BT
/F3 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Results)Tj
/F15 1 Tf
0 -1.5023 Td
(Generation)Tj
4.9417 0 Td
(and)Tj
1.7906 0 Td
(characterization)Tj
7.0252 0 Td
(of)Tj
/F16 1 Tf
1.0678 0 Td
[()]TJ
/F15 1 Tf
3.0142 0 Td
(knockout)Tj
4.1386 0 Td
(mutants)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 433.9842 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(In)Tj
1.1055 0 Td
(our)Tj
1.627 0 Td
(preliminary)Tj
4.9436 0 Td
(attempts)Tj
3.668 0 Td
(to)Tj
1.0262 0 Td
(generate)Tj
3.6056 0 Td
(CRISPR/Cas9)Tj
5.7656 0 Td
(knockout)Tj
4.0252 0 Td
(null)Tj
1.7915 0 Td
(mutants)Tj
3.5036 0 Td
(we)Tj
1.3323 0 Td
(tried)Tj
2.1033 0 Td
(the)Tj
-34.4974 -1.2982 Td
(method)Tj
3.3221 0 Td
(established)Tj
4.5978 0 Td
(for)Tj
/F9 1 Tf
1.3833 0 Td
[(\n)30()7()4()8()]TJ
/F8 1 Tf
2.4831 0 Td
(and)Tj
/F9 1 Tf
1.7348 0 Td
[(\n)30()6()11()10(\r)6()15()4()8()]TJ
4.1953 0 Td
[()9()11()2()10(\r)6()2()10()]TJ
/F8 1 Tf
3.7417 0 Td
(mosquitoes)Tj
4.7962 0 Td
([)Tj
0.83 0.64 0.02 0 k
(11)Tj
0 g
(,)Tj
0.83 0.64 0.02 0 k
1.7404 0 Td
(15)Tj
0 g
(])Tj
1.5081 0 Td
(by)Tj
1.1848 0 Td
(co-injecting)Tj
4.9946 0 Td
(syn-)Tj
-35.6822 -1.2983 Td
(thesized)Tj
3.4582 0 Td
(gRNAs)Tj
3.0897 0 Td
(\(sgRNAs\))Tj
4.1273 0 Td
(and)Tj
1.7348 0 Td
(Cas9)Tj
2.1543 0 Td
(protein)Tj
3.1521 0 Td
(into)Tj
1.8368 0 Td
(the)Tj
1.474 0 Td
(embryos)Tj
3.6624 0 Td
(of)Tj
/F9 1 Tf
1.0148 0 Td
[(\n)]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
1.0998 0 Td
[()2()16()17()11()4()6()]TJ
/F8 1 Tf
3.5149 0 Td
(G3)Tj
1.4117 0 Td
(mosquitoes.)Tj
-31.7308 -1.3039 Td
(The)Tj
1.7801 0 Td
(mosquito)Tj
4.0138 0 Td
(embryo)Tj
3.3052 0 Td
(microinjection)Tj
6.1511 0 Td
(was)Tj
1.6952 0 Td
(carried)Tj
3.0387 0 Td
(out)Tj
1.5534 0 Td
(according)Tj
4.1895 0 Td
(to)Tj
1.0262 0 Td
(established)Tj
4.5921 0 Td
(protocols)Tj
3.9628 0 Td
([)Tj
0.83 0.64 0.02 0 k
(25)Tj
0 g
()Tj
0.83 0.64 0.02 0 k
-35.3081 -1.2983 Td
(28)Tj
0 g
(].)Tj
1.7347 0 Td
(In)Tj
1.1112 0 Td
(total,)Tj
2.228 0 Td
(approximately)Tj
5.9754 0 Td
(10%)Tj
1.9276 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(eggs)Tj
1.9389 0 Td
(hatched)Tj
3.3789 0 Td
(from)Tj
2.2053 0 Td
(1000)Tj
2.126 0 Td
(micro-injected)Tj
6.1284 0 Td
(eggs,)Tj
2.16 0 Td
(and)Tj
1.7292 0 Td
(the)Tj
-35.1267 -1.2982 Td
(emerged)Tj
3.6736 0 Td
(G)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 243.8929 367.3133 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(0)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 249.6755 369.0141 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(adults)Tj
2.6135 0 Td
(were)Tj
2.1203 0 Td
(allowed)Tj
3.2881 0 Td
(to)Tj
1.0262 0 Td
(outcross)Tj
3.5773 0 Td
(with)Tj
1.9956 0 Td
(wild)Tj
1.9502 0 Td
(type)Tj
1.9105 0 Td
(mosquitoes)Tj
4.8019 0 Td
(to)Tj
1.0204 0 Td
(produce)Tj
3.5207 0 Td
(G)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 535.0677 367.3133 cm
BT
/F8 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 540.8503 369.0141 cm
BT
/F8 1 Tf
1 TL
-0.004 Tc
0 0 Td
(eggs)Tj
1.9332 0 Td
(fol-)Tj
-36.0167 -1.3039 Td
(lowed)Tj
2.6078 0 Td
(by)Tj
1.1792 0 Td
(screening)Tj
4.0536 0 Td
(for)Tj
1.3889 0 Td
(germline)Tj
3.7814 0 Td
(transmission)Tj
5.3688 0 Td
(of)Tj
1.0148 0 Td
(stable)Tj
2.4888 0 Td
(mutations,)Tj
4.5071 0 Td
(but)Tj
1.5533 0 Td
(not)Tj
1.5704 0 Td
(a)Tj
0.6577 0 Td
(single)Tj
2.5285 0 Td
(mutant)Tj
3.1463 0 Td
(was)Tj
-35.8466 -1.2982 Td
(identified)Tj
4.0592 0 Td
(in)Tj
1.0318 0 Td
(the)Tj
1.474 0 Td
(survivors')Tj
4.1102 0 Td
(progeny.)Tj
-9.479 -1.2983 Td
(We)Tj
1.6101 0 Td
(then)Tj
2.0182 0 Td
(proceeded)Tj
4.3653 0 Td
(with)Tj
2.0013 0 Td
(the)Tj
1.4683 0 Td
(development)Tj
5.4085 0 Td
(of)Tj
1.0148 0 Td
(a)Tj
0.652 0 Td
(modified)Tj
3.8494 0 Td
(method)Tj
3.3222 0 Td
(to)Tj
1.0261 0 Td
(generate)Tj
3.6113 0 Td
(CRISPR/)Tj
-31.5437 -1.2983 Td
(Cas9-induced)Tj
5.7486 0 Td
(knockout)Tj
4.0252 0 Td
(mutant)Tj
3.1464 0 Td
(in)Tj
/F9 1 Tf
1.0375 0 Td
[(\n)]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
1.0942 0 Td
[()2()16()17()11()4()6()]TJ
/F8 1 Tf
3.5206 0 Td
(mosquitoes)Tj
4.8018 0 Td
(through)Tj
3.4413 0 Td
(combination)Tj
5.3461 0 Td
(and)Tj
1.7348 0 Td
(modifi-)Tj
-33.8965 -1.3039 Td
(cation)Tj
2.6929 0 Td
(of)Tj
1.0091 0 Td
(established)Tj
4.5921 0 Td
(approaches)Tj
4.7282 0 Td
([)Tj
0.83 0.64 0.02 0 k
(16)Tj
0 g
(,)Tj
0.83 0.64 0.02 0 k
1.7348 0 Td
(28)Tj
0 g
(].)Tj
1.7291 0 Td
(We)Tj
1.6044 0 Td
(first)Tj
1.8028 0 Td
(created)Tj
3.1181 0 Td
(a)Tj
0.6463 0 Td
(transgenic)Tj
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4.3596 0 Td
[(\n)]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
1.0885 0 Td
[()2()16()17()11()4()6()]TJ
/F8 1 Tf
3.515 0 Td
(line)Tj
1.7007 0 Td
(express-)Tj
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(ing)Tj
1.5023 0 Td
(three)Tj
2.2621 0 Td
(gRNAs)Tj
3.0897 0 Td
(targeting)Tj
3.7871 0 Td
(the)Tj
/F9 1 Tf
1.474 0 Td
[()]TJ
/F8 1 Tf
2.8856 0 Td
(gene)Tj
2.0807 0 Td
(\()Tj
0.83 0.64 0.02 0 k
(S1)Tj
1.5136 0 Td
(Fig)Tj
0 g
(\).)Tj
2.0466 0 Td
(All)Tj
1.3947 0 Td
(three)Tj
/F9 1 Tf
2.2677 0 Td
[()]TJ
/F8 1 Tf
(-targeting)Tj
6.8088 0 Td
(gRNAs)Tj
3.0896 0 Td
(were)Tj
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(driven)Tj
2.8119 0 Td
(by)Tj
1.1792 0 Td
(the)Tj
/F9 1 Tf
1.474 0 Td
[(\n)]TJ
/F8 1 Tf
(.)Tj
/F9 1 Tf
1.0942 0 Td
[()2()16()17()11()4()6()]TJ
/F8 1 Tf
3.5206 0 Td
(U6)Tj
1.4287 0 Td
(snRNA)Tj
3.1748 0 Td
(polymerase)Tj
4.7791 0 Td
(III)Tj
1.2359 0 Td
(promoter)Tj
4.0365 0 Td
(\(AGAP013557\).)Tj
6.6217 0 Td
(The)Tj
1.7859 0 Td
(specific)Tj
-33.1425 -1.3039 Td
(sequences)Tj
4.2179 0 Td
(of)Tj
1.0148 0 Td
(individual)Tj
4.286 0 Td
(gRNAs)Tj
3.0897 0 Td
(were)Tj
2.1203 0 Td
(synthesized)Tj
4.8302 0 Td
(as)Tj
1.0148 0 Td
(short)Tj
2.2847 0 Td
(DNA)Tj
2.3754 0 Td
(linkers)Tj
2.9367 0 Td
(\()Tj
0.83 0.64 0.02 0 k
(S1)Tj
1.508 0 Td
(Table)Tj
0 g
(\))Tj
2.7836 0 Td
(and)Tj
1.7291 0 Td
(sepa-)Tj
-34.1912 -1.2983 Td
(rately)Tj
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(cloned)Tj
2.897 0 Td
(into)Tj
1.8368 0 Td
(plasmid)Tj
3.3959 0 Td
(modules)Tj
3.634 0 Td
(carrying)Tj
3.5546 0 Td
(the)Tj
1.4684 0 Td
(U6)Tj
1.4343 0 Td
(promoter)Tj
4.0308 0 Td
(sequence)Tj
3.8608 0 Td
(followed)Tj
3.651 0 Td
(by)Tj
1.1792 0 Td
(a)Tj
0.652 0 Td
(gRNA-)Tj
-34.0439 -1.2983 Td
(expression)Tj
4.4617 0 Td
(template,)Tj
3.9061 0 Td
(and)Tj
1.7348 0 Td
(assembled)Tj
4.32 0 Td
(by)Tj
1.1848 0 Td
(a)Tj
0.652 0 Td
(Golden)Tj
3.1861 0 Td
(Gate)Tj
2.092 0 Td
(cloning)Tj
3.2258 0 Td
(reaction)Tj
3.4979 0 Td
(into)Tj
1.8369 0 Td
(the)Tj
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