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Supplementary Information For

“Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination”

Daniel J. Dickinson1,3, Jordan D. Ward4, David J. Reiner2,3,5 and Bob Goldstein1,3

1Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 4Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; 5Present address: Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX USA. Correspondence: Daniel J. Dickinson Department of Biology University of North Carolina at Chapel Hill Chapel Hill, NC 27599-3280 Tel: 650-815-1923 Email: [email protected]

Contents Supplementary Figure 1: Multiple alignment of C. elegans U6 genes 2

Supplementary Figure 2: Workflow and timetable 5

Supplementary Table 1: Efficiency of homologous recombination 7

Supplementary Table 2: Scoring of Pvl phenotype in lin-31 mutants 8

Supplementary Table 3: Candidate off-target cleavage sites sequenced 9

Supplementary Table 4: C. elegans strains used in this study 10

Supplementary Table 5: sgRNA targeting sequences used in this study 11

Supplementary Table 6: Primers used in this study 12

Supplementary Protocol for Cas9-triggered homologous recombination 14

Nature Methods: doi:10.1038/nmeth.2641

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2Nature Methods: doi:10.1038/nmeth.2641

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474599688158343512860931899108

910 920 930 940 950 960 970 980 990

A TTA TTAAAAAAAC - - - - - - - - - - - - - - - - - - - - - - - - - ACC TTTTTTCCAAAA TTA TTCCA - - - - - - - - - - - - - - - - - CAAAAAA TA TG TTA TGA - - - -A TTTTCAAG TC TAC TTTGAAA TA TTTCAAG TA TTTTCGAAC TCC TTTTCCAA TTTTGAA TAA - - - - - - - - - - - - - - - - - C TC TAAACA TTTTAAGAG TTCA TG TAGAACGAACACA TAAAAAA TTA TTAA TA TC - - - - GA TTCA TTGGC TAAAAAG TTA TTG - - - - - - - - - - - - - - - - - ACGGAAA TAAG TA TCAAG TTTAGA TC TAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TTTA TG TTT - - - - - - - - - - - TGG - - - - - - - - - - - - - - - - - TTTCCG TC TACCCG TCAC TA TAA TTC TAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A TTTTGCA TGAAACACAGG TTCA - - - - - - - - - - - - - - - - - A TTAGA TTTAG TGA TTTG TACTTG TT - AA TGACAG - - - - - CA TTTTAAGAGC T - CCCAACACA TAG TG TTT - - - - - - - - - - - - - - - - - - - - - - - - CCAA TG TTA TACCCAA TCAA TAA TAGTT - TC - A TTTACAGACC TTCC TTTTGAGACC T - TCCAACAC TGAG TG TTT - - - - - - - - - - - - - - - - - - - - - - - - CCAA TG TTA TACCCAA TCAA TAA TC TA TG TT - TC TTAAA - - - - TGCA TTTTTAGAGC T - CCCAACACA TAG TG TTT - - - - - - - - - - - - - - - - - - - - - - - - CCAA TG TTA TACCCAA TCAA TAA TAGG TGCAGAAAGAG TGAA TGACA TA TTTGGAG TTA TG TAAGACAC TA TGAC TA TGC TAGGGAAG TGGAGA TGCAAA TCGA TA TTGG TA TCGC TA TAGAA TAG- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CCC TCC TC TTT - - - - - - - - - - - - - - - - - - - - - - - - - - - - TCCCAGCC TCCA TC TC TGCC TC

475600689159344513861932900109

5706997862494356079551026999194

1010 1020 1030 1040 1050 1060 1070 1080 1090

- - - - AA TGCC TACACCC TC TCACACACAC TC TTTA TAC TAC TC TG TCAAAC TCACGAGA TG TC TGCCGCC TC TTG TG TCCCCCC TA TA TAAACACC TCC TTTGCAAGAGC TCCACCC TTAA TGG TA TGCAAAACA TAGAGGA TGACAAAACCCGCAAGACG TC TGCCGCC TC TTG TG TCCCCCC TA TA TAAACACC TCC TTGG TTCGA TTC TTGCCC TTTTTGAG TTGC TTGACGGAGAA - - TTGCGGAACCCACGAGA TG TC TGCCGCC TTGCAGG TGGCCAC TA TA TAAGCAACAAC TTCA TCCG TCC TTC - CCGCC - - - - - - - - ACCAAAAA TAG TG TG TAGCGGAACCCGGGA TG TG TCG TG TAC TG TCCAAA TGGCCC TTA TA TAAACAACCCG TAAGACCG TC TAACACCACC - - - - - - - - ACCAA TAA TAA TG TG TGGCGGAACCCGGGAAG TG TCG TG TAC TA TCCAAA TAGCCC TTA TA TAAACGACCCG T- - - - - CAAG TCAA TAAAC TACC TC TACAC TA TTTC TGG TAA TAGGCGAAACC TC TACAC TG TCAG TCAC TTTG TAAGG TG TGCC TA TG TA TTTCCA TAA T- - - - - CAAG TCAA TAAAC TACC TC TG TC TTA TTTTTGG TAA TAGGCGAAACC TC TACAC TG TCAG TCAC TTTG TAAGG TG TGCC TA TA TAA TTCCA TAA T- - - - - CAAG TCAA TAAAC TACC TC TACAC TA TTTC TGG TAA TAGGCGAAACC TC TACAC TG TCAG TCAC TTTG TAAGG TG TGCC TA TA TA TTTCCA TAA TG TGCACAAA TTA TTGA TC TACA TC TACA TTTGG TA TAG TG TC TTACA TAAC TCCAAA TA TG TCAGC TGC TG TG TAAA TGG TCCC TA TA TAA TTGAA TTGCC TCCCCCAACAGCC TC TCC - - - - - - - - - - - - - - CA TAGCCCCCAGCGGAACCCA TGACA TG TC TGC TACAC TCC TTAC TGCCCC TA TA TAAACAGCCCC T

571700787250436608956

10271000

195

670799886349535706105411251098294

1110 1120 1130 1140 1150 1160 1170 1180 1190

A TTGCGAGA TG TC TTG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGA TTGCGAGA TG TC TCG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGGG TCC TGGC TG TC TCG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGC TTTCGA TAAA TC TCG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGTTTTCCA TAAA TC TCG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGA TTTC - A TACAAA TTG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGA TTTC - A TACAAA TCG TTC TTCCAAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGA TTTC - A TACAAA TTG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGAAA TC - TAAA TG TTTG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TGACCCGC TAG TA TTTCG TTC TTCCGAGAACA TA TAC TAAAA TTGGAACAA TACAGAGAAGA TTAGCA TGGCCCC TGCGCAAGGA TGACACGCAAA TTCG TG

671800887350536707

105511261099

295

697826986383569744109211631139327

1210 1220 1230 1240 1250 1260 1270 1280 1290

AAGCG TTCCAAA TTTTTTG TGAAA TTT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTG TGAAA TTT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTTTAAAA TTTCGCG TAACC TACCGGAAGC TCG TAAAA TTTTTGCC TTTTGAAGA TTTTACGAC TGA TCC TC TAA TG TACAGAGAAGCG TTCCAAA TTTTTTG TGAAA TTTTTGAAAG - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTG TGAAA TTTTGAA TGA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTGAC TAA TTTC TGG TA - - - TTA TT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTGAC TAA TTTC TGACA - - - TAGAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTGAC TAA TTTC TGACA - - - TAGAG - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTTTTCGA TTTTTCCAAAAACCA TAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGCG TTCCAAA TTTTTCGACAAA TTTTGAA TG - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

698827987384570745

109311641140

328

7178371086438625751110611861146360

1310 1320 1330 1340 1350 1360 1370 1380 1390

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TC TA TAA TTTTTTTTGCAGA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TTCG TG TTTC - - - - - - - - - ATAA TAA TTCG TTTG TCC TGAA TCAA TCAA TTCACAAAGAAG TCGAGAC TTTTA TGAG TCG TCC TG TAGG TG TC TA TTTC TTTCAGAA TTC TTTTTCAA TA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TTGA TA TGCAG - A TTGA TTGAA TAAAGC TCAAC TA TTA TGG TAC TTTA TTA T - AC TG- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GGAAAA TGCGG TACCAAA TTTGAAAA TTTCAAA TTG TG TAG T - TTG TA TGA TGACCC- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G TTTAGA - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TTTTACA TGACA - - - - - - - - - AG- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TTTTACA TA TA TC TTC TC TG TA T- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TTTCAGA - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CC TGCC TGCC TTTACC TAG TTTTAAAAAA TTG T - - - - - - - - - -

718838

1087439626752

110711871147

7198391186499686796118612861199

1410 1420 1430 1440 1450 1460 1470 1480 1490

AA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -GA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -TA TG TGAAAAA TAGA TAAA TACAACACG TA TA TTAAGAGCAA TAAACA TTTTTCAAAAAAAAAAGCAAAAGGAACAAAAA TTACAGGGAAAGCGAAAA TTAG TACAAAGG TAAG - - - - - - - - - - - - TAA TTA TTCGCA TTAA TTAAC TGACA TTACGGA - - AA TTCAAAC - - - - - - - - - - - - - - - - - - - - - - - - AAA TT -AGCA TTG TTGGAAG - - - - - - - - - - - - - A TTTA TCAAC - - TCGCGGA TTCAAA TCGCGGA TCAAA TCCAA T - - - - - - - - - - - - - - - - - - - - - - - - AC TTTT- - - - - - A TTG TAAGAAAA TTTT - - - - - - - - - - - - - - - - - - - - - ACA TTCCAGA - - - - - - - AAA TG TA TC - - - - - - - - - - - CAAAAAAC T - - - - - - - - - - TAAAG TCGG TG TAAC TAAA TTTTTTC TA TA - - - - - - - - - - TTTAAC TTACCAG TTC TTC TTGAA TAAA TC TG TGG TC TTC TCAGAA TAG T - - - - - - - - - - TTG TTTCAA TTTTAG TA TA TG TTC TCGAAAGAAA TAC TC TTAAAA TAA TC TAA TTTTACCAAGAGCCA TAA TTCAACAC TTCAAAAAG TTAG TAG TTG TTC- - - - - - - - - - TCAGAAGACGC TC - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - AAG TAAGAA TTTG TGC TC TTGAACAA TG TG TTGA TGGAAA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

1187

687797

118712871200

720840120050070080012001300

1510

- - - - - - - - - - - - - A- - - - - - - - - - - - - ACAAGAAA TTTA TGA- - - - - - - - - - - - - ATGA TTTAA TTGGGGACG - - - - - - - - - - CCGAAC TTCGA TA TCTAAA TTG TCAGC TAT - - - - - - - - - - - - -- - - - - - - - - - - - - -

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

R07E5.16C28A5.7F54D8.7

Y17G9A.85T08B6.62F54C8.8

F54C8.10F54C8.9

K09B11.11W05B2.8

U6 snRNA


danieldickinson

Typewritten Text

danieldickinson

Typewritten Text

Supplementary Fig. 1, con't

Supplementary Figure 1: Multiple alignment of C. elegans U6 genes

Residues that are >50% conserved are highlighted, and the U6 RNA sequence is indicated

above the alignment. The alignment was produced using CLC Sequence Viewer software.


danieldickinson

Typewritten Text

Before Experiment:Generate plasmids

Day 0: Inject

Day 9: Heat shockto kill animals with arrays

Day 10: Pick candidateknock-in animals

Day 15: Isolate genomic DNAand confirm knock-in by PCR

Day 0: Inject

Day 2: Pick fluorescent F1’s

Days 4-7: Pick Unc animals

Isol

atio

n of

kno

ck-in

alle

les

(2-3

wee

ks)

Rem

oval

of s

elec

tabl

e m

arke

r (op

tiona

l; 1

wee

k)

Cas9+sgRNAplasmid(8.1 kb)

Peft-3Cas9

tbb-23’UTR

PU6sgRNA

AmpR

+gfp

unc-119(+)

5’ homology 3’ homology3’UTR

Cas9 + sgRNA plasmid:Insert targeting sequence using

site-directed mutagenesis

Homologous repair tempate:1) Clone 3-4 kb genomic fragment

2) Modify as desired and insert selectable marker

Injection mix:- Cas9+sgRNA plasmid- Homologous repair template- PEEL-1 negative selection construct- Fluorescent co-injection markers

Inject 50-80 wormsper experiment

25°C until starvation

Mixture of knock-inand array animals

34°C, 4h

Array animals are killedby heat shock- induced PEEL-1

Pick non-UncPEEL-1 survivorslacking fluorescent co-injection markers

gfp unc-119(+)LoxP LoxP

1: Upstream 3: unc-119 3’2: GFP R 4: Downstream

Injection mix:- Peft-3::Cre plasmid- Fluorescent co-injection marker

Inject 15-20 wormsper experiment

25°C

Single 10-20 transgenicprogeny to new plates

25°C

At 25°C, unc-119 mutantsare slightly Dpy and Egl in addition to Unc


danieldickinson

Typewritten Text

Supplementary Figure 2

Supplementary Figure 2: Workflow and timetable for modifying the C. elegans

genome using Cas9-triggered homologous recombination

The basic steps in the procedure are shown, using the nmy-2::gfp knock-in experiment as an

example. Please refer to the Supplementary Text for a detailed protocol.


danieldickinson

Typewritten Text

Transgene DSB Method Total Injections Successful Injections Transgene Insertions Insertion EfficiencyPmex-5::4xYPET::tbb-2 3'UTR Mos1 55 17 6 35%Pmex-5::GFP::tbb-2 3'UTR Mos1 54 27 1 4%Phis-72::GFP::tbb-2 3'UTR Mos1 64 26 4 15%Total (Mos1) Mos1 173 70 11 16%

Pmex-5::4xYPET::tbb-2 3'UTR Cas9 66 13 2 15%Pmex-5::GFP::tbb-2 3'UTR Cas9 79 37 12 32%Phis-72::GFP::tbb-2 3'UTR Cas9 72 22 2 9%Total (Cas9) Cas9 217 72 16 22%

Supplementary Table 1: Efficiency of MosSCI and Cas9-triggered homologous recombination for single-copy transgene insertionThis table presents the raw data depicted in Fig. 2b. “Total injections” is the number of animals that survived injection. “Successful injections” is the number of animals that produced non-Unc progeny (which could be due to either single-copy transgene insertion or extrachromosomal array formation). “Transgene insertions” is the number of animals that produced progeny carrying single copy integrated transgenes and lacking extrachromosomal array markers.


Genotype Number Scored Number Pvl Percent PvlWild Type (N2) 406 1 0.2%

lin-31(cp1[4T->A]) unc-119(+) II; unc-119(ed3) III 114 59 52%lin-31(cp2[4T->A]) unc-119(+) II; unc-119(ed3) III 153 86 56%Total lin-31(4T->A) 267 145 54%

lin-31(cp3[4T->E]) unc-119(+) II; unc-119(ed3) III 48 22 46%lin-31(cp4[4T->E]) unc-119(+) II; unc-119(ed3) III 54 24 44%lin-31(cp5[4T->E]) unc-119(+) II; unc-119(ed3) III 115 59 51%Total lin-31(4T->E) 217 105 48%

Supplementary Table 2: Scoring of Pvl phenotype in lin-31 mutants This table presents the raw data depicted in Fig. 5e.


Target Sequence # of Mismatches Mutations?nmy-2 (3' end) GTTAGTTGCGAACTGAGTCGCGG 0Between inx-2 and F08G12.2 AGTACTGTCATACTGAGTCGAGG 6 None Detectedsex-1 GCTAAGAGCTTACTGAGTCGCGG 6 None DetectedBetween F07A5.4 and sue-1 TATCATTTCGAACCGAGTCGGGG 6 None DetectedC30F12.2 ATCATACGAGAACTGAATCGTGG 7 None DetectedBetween Y102A5C.2 and Y102A5C.4 TGTCGACACGAAATGAGTCGCGG 7 None Detectednhr-211 CATTTTCCGGAACTGGGTCGGGG 8 None DetectedE01G6.1 GAAATACACTTACTGAGTCGAGG 8 None DetectedBetween F58H10.1 and nsf-1 CTGAAAAAGATACTGAGTCGAGG 9 None Detectedceh-89 AAGGAATCGGAACTGTGTCGCGG 9 None Detectedlin-24 TTAGTGACTGAACTGAGTGGCGG 9 None Detected

lin-31 (5' end of last exon) GAGAGATTTTCGATGGAGCCGGG 0Y51H1A.2 CAGATCGGTGCGATGGAGCCCGG 6 None DetectedBetween F15A4.10 and T21B4.15 GGAATAAAAAGGATGGAGCCTGG 8 None Detectednhl-3 TTTTGTTGTATGATGGAGCCGGG 8 None DetectedY57A10A.7 TTGCTGAACTTGATGGAGCCGGG 9 None Detected

Supplementary Table 3: Candidate off-target cleavage sites sequencedThis table lists the putative off-target cleavage sites for Cas9 with our sgRNAs that we sequenced to look for off-target mutations. Mismatches to the sgRNA sequence are shown in red, and the PAM is underlined.


Strain Name Genotype ReferenceDP38 unc-119(ed3) III 43EG6699 ttTi5605 II; unc-119(ed3) III 17JJ1473 unc-119(ed3) III; zuIs45[Pnmy-2::NMY-2::GFP + unc-119(+)] V 27LP116 cpSi4[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP117 cpSi5[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP118 cpSi6[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP119 cpSi7[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP120 cpSi8[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP121 cpSi9[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP122 cpIs5[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP123 cpIs6[Pmex-5::4xYPET::3xFlag::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP126 lin-31(cp1[T145A T200A T218A T220A + LoxP unc-119(+) LoxP]) II; unc-119(ed3) III This StudyLP127 lin-31(cp2[T145A T200A T218A T220A + LoxP unc-119(+) LoxP]) II; unc-119(ed3) III This StudyLP128 lin-31(cp3[T145E T200E T218E T220E + LoxP unc-119(+) LoxP]) II; unc-119(ed3) III This StudyLP129 lin-31(cp4[T145E T200E T218E T220E + LoxP unc-119(+) LoxP]) II; unc-119(ed3) III This StudyLP130 lin-31(cp5[T145E T200E T218E T220E + LoxP unc-119(+) LoxP]) II; unc-119(ed3) III This StudyLP132 nmy-2(cp7[nmy-2::gfp + LoxP unc-119(+) LoxP]) I; unc-119(ed3) III This StudyLP133 nmy-2(cp7[nmy-2::gfp + LoxP unc-119(+) LoxP]) I; unc-119(ed3) III This StudyLP134 nmy-2(cp7[nmy-2::gfp + LoxP unc-119(+) LoxP]) I; unc-119(ed3) III This StudyLP135 cpSi10[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP136 cpIs10[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP137 cpIs11[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP138 cpIs12[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP139 cpIs13[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP140 cpIs14[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP141 cpIs15[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II;; unc-119(ed3) III This StudyLP142 cpIs16[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP143 cpIs17[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP144 cpIs18[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP145 cpIs19[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP146 cpIs20[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP147 cpIs21[Pmex-5::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP148 unc-119(ed3) his-72(cp10[his-72::gfp+ LoxP unc-119(+) LoxP]) III This StudyLP149 cpSi11[Phis-72::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP150 cpSi12[Phis-72::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP151 cpSi13[Phis-72::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP152 cpSi14[Phis-72::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP153 cpIs23[Phis-72::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP154 cpIs24[Phis-72::GFP::tbb-2 3'UTR + unc-119(+)] II; unc-119(ed3) III This StudyLP157 lin-31(cp11[T145A T200A T218A T220A + LoxP]) II; unc-119(ed3) III This StudyLP158 lin-31(cp12[T145E T200E T218E T220E + LoxP]) II; unc-119(ed3) III This StudyLP159 nmy-2(cp13[nmy-2::gfp + LoxP]) I; unc-119(ed3) III This Study

Supplementary Table 4: C. elegans strains used in this study


Target Genomic Sequence sgRNA targeting sequenceChromosome II near ttTi5605 Mos1 insertion site GATATCAGTCTGTTTCGTAACGG GATATCAGTCTGTTTCGTAAnmy-2 (3' end) GTTAGTTGCGAACTGAGTCGCGG GTTAGTTGCGAACTGAGTCGhis-72 (3' end) GAGCTTAAGCACGTTCTCCGCGG GAGCTTAAGCACGTTCTCCGlin-31 (5' end of last exon) GAGAGATTTTCGATGGAGCCGGG GAGAGATTTTCGATGGAGCC

Supplementary Table 5: sgRNA targeting sequences used in this studyThe PAM is underlined.


Primer Description Primer SequenceCas9-sgRNA plasmidCas9 For TCAGTTGGGAAACACTTTGCTCas9 Rev GCTTGAAAGGATTTTGCATTTATCPeft-3 Rev (Vector) TTTTTCTAGAGCAAAGTGTTTCCCAACtbb-2 3'UTR For (Vector) ctaaactagtGATAAATGCAAAATCCTTTCAAGCPU6::sgRNA (Synthetic gBlock fragment)sgRNA vector Rev GTTTTCCCAGTCACGACGTTsgRNA vector For CTGGCGTAATAGCGAAGAGG

Deletion of the Cas9 target from MosSCI constructspCFJ150 Δtarget For CCGTATCACAACTGAGAAAAAAGtbb-2 3'UTR Rev gcatcgcgcgcaccgtacgTGAGACTTTTTTCTTGGCGG

PCR genotyping of the ttTi5605 locusttTi5605 Upstream AGGCAGAATGTGAACAAGACTCGttTi5605 Downstream ATCGGGAGGCGAACCTAACTGunc-119(+) 5' out CAATTCATCCCGGTTTCTGTunc-119(+) 3' out TTCGCTGTCCTGTCACACTC

nmy-2::gfp repair templatenmy-2 5' arm For TCTCAAACTAGAGATTGACAATTTGGnmy-2 5' arm Rev tcctttactcatggctccgctagctcctgagttacggacagagtctctaTCTTCATCGGTGTTGCCGFP For tcaggagctagcggagccATGAGTAAAGGAGAAGAACTTTTCACTGGFP Rev tctcttgtcatcgtcatccttgtaatcggatccggatccacgcttatcgtcatcgtccttgtagtcgcatgcTTTGTATAGTTCATCCATGCCATGnmy-2 3'UTR For gattacaaggatgacgatgacaagagaggatctggatctgactacaaggacgatgacgataagcgttaaCCTCTAAAGTTTTTAATTCCGGAnmy-2 3'UTR Rev TGCGGACATGTCAATTTGTnmy-2::LoxP::unc-119(+) For tatttaatgtaacaaattgacatgtccgcaataacttcgtatagcatacattatacgaagttatCTTTGAGCCAATTTATCCAAGnmy-2::LoxP::unc-119(+) Rev ataatccggaattaaaaactttagaggttaataacttcgtataatgtatgctatacgaagttatCCTAGTTCTAGACATTCTCTAATGnmy-2 3' arm For TAACCTCTAAAGTTTTTAATTCCGGATnmy-2 3' arm Rev TGACAAGGACATTCTCGGA

Cas9-sgRNA plasmid targeting nmy-2nmy-2 sgRNA For ttagttgcgaactgagtcgGTTTTAGAGCTAGAAATAGCAAGTsgRNA Rev CAAGACATCTCGCAATAGG

PCR genotyping of the nmy-2 locusnmy-2 Upstream TGACGCACAAGAAAAGATCGnmy-2 Downstream ATCAACAAAACATGCGTCCAGFP Rev TTTGTATAGTTCATCCATGCCATGunc-119(+) 3' out TTCGCTGTCCTGTCACACTC

his-72::gfp repair templatehis-72 5' arm For GACCCCACAAAATCGATACGhis-72 5' arm Rev tcctttactcatggctccgctagctcctgagGCACGTTCTCCtctGATGCGTCTGGCGAGGFP For tcaggagctagcggagccATGAGTAAAGGAGAAGAACTTTTCACTGGFP Rev tctcttgtcatcgtcatccttgtaatcggatccggatccacgcttatcgtcatcgtccttgtagtcgcatgcTTTGTATAGTTCATCCATGCCATGhis-72 3'UTR For gattacaaggatgacgatgacaagagaggatctggatctgactacaaggacgatgacgataagcgttaaGCTCCATCACCAATTCTCGhis-72 3'UTR Rev ttacaaggacttggataaattggctcaaagataacttcgtataatgtatgctatacgaagttatGCGGCGTGGAATATAGTTunc-119(+) For CTTTGAGCCAATTTATCCAAGTChis-72::LoxP::unc-119(+) Rev taaaagtgcttcgagaattggtgatggagcataacttcgtataatgtatgctatacgaagttatCCTAGTTCTAGACATTCTCTAATGhis-72 3' arm For GCTCCATCACCAATTCTCGAhis-72 3' arm Rev AGGCTGAATTTGCTCTGAG

Cas9-sgRNA plasmid targeting his-72his-72 sgRNA For agcttaagcacgttctccgGTTTTAGAGCTAGAAATAGCAAGTsgRNA Rev CAAGACATCTCGCAATAGG

PCR genotyping of the his-72 locushis-72 Upstream GTGGATTCAGGATCCTTCGhis-72 Downstream TTTTGAGCTGAAGCTTGTATGGGFP Rev TTTGTATAGTTCATCCATGCCATGunc-119(+) 3' out TTCGCTGTCCTGTCACACTC


Supplementary Table 6: Primers used in this studyPrimers are listed for each construct. For each primer, the region that anneals to the target is capitalized. LoxP sites are underlined.

lin-31(4T->A) and lin-31(4T->E) repair templateslin-31 5' arm For ATTTGGTTCAGCCGAAATTGlin-31 5' arm Rev gggagcagtggaagaggatttttgcgagagattttgcttggggctggaTGGTGGAAGTCGTCGTlin-31 4T->A 3' end (Synthetic gBlock fragment)lin-31 4T->E 3' end (Synthetic gBlock fragment)lin-31 3'UTR For CAAATGCGCTCTGTTAACClin-31 3'UTR Rev ttacaaggacttggataaattggctcaaagataacttcgtataatgtatgctatacgaagttatAAAAACGTTATTTGGTTGTTTATGACTAAunc-119(+) For CTTTGAGCCAATTTATCCAAGTClin-31::LoxP::unc-119(+) Rev actggcaactttttcattatttggagaaaaataacttcgtataatgtatgctatacgaagttatCCTAGTTCTAGACATTCTCTAATGAAAAlin-31 3' arm For TTTTCTCCAAATAATGAAAAAGTTGCClin-31 3' arm Rev GTCGAACCCAGAAATCAAC

Cas9-sgRNA plasmid targeting lin-31lin-31 sgRNA For agagattttcgatggagccGTTTTAGAGCTAGAAATAGCAAGTsgRNA Rev CAAGACATCTCGCAATAGG

PCR genotyping and sequencing of the lin-31 locuslin-31 Upstream GCTCAGGGTTAGGCTTAGGGlin-31 Downstream ATATGTGGCACTCGCTTCGunc-119(+) 5' out CAATTCATCCCGGTTTCTGTunc-119(+) 3' out TTCGCTGTCCTGTCACACTClin-31 Seq CGGGATGTCTCTGTCTCTTCAACT

Cloning of Cre recombinase into pDONR221Cre For attB1 ggggacaagtttgtacaaaaaagcaggctCCATGGGCGCACCCCre Rev attB2 ggggaccactttgtacaagaaagctgggtaCTAATCGCCATCTTCCAGCA


Supplementary Protocol: Cas9-triggered homologous recombination We have provided the following protocol in order to facilitate rapid adoption of our

methodology by members of the C. elegans research community. Supplementary Fig. 2 shows a

graphical representation of the steps in this procedure and the time required. All necessary plasmids

are available from Addgene.

Construct Design and Cloning

Homologous repair template

We describe our cloning strategy in general terms, but this can be substituted with another approach

if desired.

• Design the desired genomic modification and make a sketch of the modified locus. Using a

DNA sequence editor, generate a file that contains the sequence of the locus in its desired final

state, following the guidelines below and using Figs. 3a and 5b as examples.

• PCR-amplify a 3–4 kb genomic region, centered on the desired modification, from N2 genomic

DNA. We aim to have ~1.5 kb of unmodified sequence at each end of our homologous repair

template to allow homologous recombination. Clone the genomic fragment into any suitable

vector (we use the ZeroBlunt TOPO kit from Life Technologies).

• Build the modifications you want into your cloned genomic fragment. The exact steps will vary

depending on the genome modification you want to make, but for GFP knock-ins we insert

GFP between the last amino acid codon of the gene and the stop codon, and the unc-119(+)

rescue gene downstream of the 3’UTR. We have only tested unc-119 selection, but other

selectable markers could likely be substituted. If you wish to remove the selectable marker later,

flank it with LoxP sites (5’-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3’). We

have built our homologous repair templates using Gibson Assembly Master Mix from NEB, but

other cloning methods could be used instead.

• IMPORTANT: Ensure that the Cas9 target sequence (see below) is NOT present in the

homologous repair template. Otherwise, Cas9 will cleave your repair template in addition to the

genomic DNA, and recombination efficiency will suffer. If the targeting sequence is part of a

coding region then you will need to introduce silent mutations to block cleavage. If possible, the

simplest and most effective approach is to mutate the PAM (NGG motif), since this motif is


absolutely required for cleavage of a substrate by Cas9. If a PAM mutation is not feasible,

introduce at least 5–6 mutations in the target sequence. Mutations closer to the 3’ end of the

targeting sequence are more likely to prevent cleavage.

Choose the Cas9 target site

• Target sites conform to the pattern 5’G-N19-NGG-3’, where N is any base. The initial G is

required for transcription by the U6 promoter, and the NGG motif is required for Cas9

cleavage. Locate a target sequence near the site you wish to modify.

• IMPORTANT: In general, the Cas9 target sequence and the selectable marker should flank the

desired genome modifications. The presence of these two landmarks fixes the location of the

homologous recombination to the homology arms at the ends of the repair template, ensuring

that all of the isolated recombinants will contain the desired modifications. For example, for C-

terminal GFP insertions, we use a Cas9 target sequence at or near the 3’ end of the coding

sequence, upstream of the 3’UTR, and place the unc-119(+) cassette downstream of the 3’UTR

in the repair template (see Fig. 3a for an example). The desired modification (GFP insertion) lies

between the Cas9 target site and the unc-119(+) marker. For point mutations, the target

sequence should be located at or near the 5’ end of the region containing the desired

mutation(s), and the unc-119(+) cassette should be 3’ of the desired mutations in the repair

template (see Fig. 5b for an example).

• Aside from these general constraints, the target sequence may be oriented on either strand, and

its exact location relative to the site to be modified is not critical. GN19-NGG motifs occur

every 32 bases in random sequence, and we were able to find target sites matching the GN19-

NGG sequence requirement within 50–100 bp of the desired insertion site (and much closer in

most cases) for all of our homologous recombination experiments.

• At this stage, it is advisable to check the uniqueness of the target sequence by BLASTing against

the C. elegans genome. We recommend avoiding targeting sequences with fewer than 5–6

mismatches to the next most similar site in the genome. Remember that if a putative off-target

site has high homology to the GN19 targeting sequence you choose, the N in the NGG motif

does not have to match the target in order to be cleaved: for example, if your target sequence is

GAAAACCCCCGGGGGTTTTT-AGG, then the sequence

GAAAACCCCCGGGGGTTTTT-CGG would also be cleaved. Although Cas9 can cleave

off-target sites under some conditions32, the determinants of its specificity are still not


completely understood. We therefore keep a list of the closest matches for each targeting

sequence we design, to facilitate later sequencing for identification of any off-target mutations.

Although we found no evidence for a high non-specific activity of Cas9 in C. elegans, we

advocate isolating multiple lines for each genomic modification, sequencing of candidate off-

target loci, and outcrossing as prudent measures to be adopted until the factors governing Cas9

specificity are more completely understood.

Cas9-sgRNA construct

• We use NEB’s Q5 Site-Directed Mutagenesis Kit to insert the targeting sequence into our Cas9-

sgRNA construct (Addgene #47549). Use forward primer 5’-

N19GTTTTAGAGCTAGAAATAGCAAGT-3’, where N19 is replaced by the desired 19 bp

targeting sequence, and reverse primer 5’-CAAGACATCTCGCAATAGG-3’. Note that the

initial G in the GN19 sequence is included in the reverse primer.

• IMPORTANT: Do not include the PAM (NGG motif) in your primers for the Cas9-sgRNA

construct. The NGG motif must be present in the target DNA, but it is not part of the sgRNA

(Fig. 1a).

Injection and Selection

Our selection protocol is based on, and is highly similar to, the MosSCI protocol developed

by Christian Frøkjær-Jensen and colleagues17. We are greatly indebted to Dr. Frøkjær-Jensen for

developing such an effective procedure and for sharing advice and reagents.

• Obtain worms of an appropriate strain for injection. Although we used strain DP38 (unc-

119(ed3) III) for initial experiments, we have subsequently switched to the outcrossed derivative

HT1593. HT1593 has a slightly weaker phenotype than DP38 (HT1593 animals are less Dpy

and more active), but this has not affected our ability to recognize plates with rescued animals or

isolate recombinants. Therefore we recommend that others use HT1593 for unc-119 selection

experiments. In principle, any strain compatible with your selectable marker can be substituted.

Our selection procedure is also optimized for unc-119 selection, and the use of other selectable

markers may require subtle changes. If using unc-119 selection, note that unc-119 worms are

healthier and easier to inject when grown on HB101 bacteria at 15°C.

• The night before injection, pick ~80 L4 animals to a fresh plate and allow them to mature into

adults at 15°C overnight.


Day 0: Injection

• Prepare an injection mix containing the following plasmids:

o 10 ng/µL homologous repair template

o 50 ng/µL Cas9-sgRNA construct with your targeting sequence

o 10 ng/µL pMA122 (heat-shock driven PEEL-1 negative selection; Addgene #34873)

o 10 ng/µL pGH8 (Prab-8::mCherry neuronal co-injection marker; Addgene #19359)

o 5 ng/µL pCFJ104 (Pmyo-3::mCherry body wall muscle co-injection marker; Addgene

#19328)

o 2.5 ng/µL pCFJ90 (Pmyo-2::mCherry pharyngeal co-injection marker; Addgene #19327)

Miniprep-quality DNA is sufficient, and we do not linearize plasmids prior to injection.

• Inject the mixture into the gonads of the young adult worms.

• Transfer the injected worms to new seeded plates (three animals per plate works well in our

hands).

• Put the plates at 25°C until the food bacteria are consumed and the Unc worms make piles

(these are visible to the naked eye as big clumps of worms on the plate, where the edge of the

bacterial lawn used to be).

Day 7–9: Heat Shock

• Examine the plates and identify those that contain unc-119 rescued (i.e. normally moving)

animals. In our experience, plates with rescues are obvious because they contain many crawling

worms. You should not have to spend a lot of time searching to spot rescued worms. The

number of plates with rescued animals will vary depending on your injection experience, but is

usually greater than half in our hands.

• Heat shock rescued plates at 34°C for 4 hours in an air incubator. This activates expression of

the PEEL-1 toxin, which kills animals that carry extrachromosomal arrays. After heat shock,

return the plates to 25°C overnight to allow the array-carrying animals to die.

Day 8–10: Pick Candidate knock-in animals

• The day after heat shock, examine the plates for normally moving animals that survived heat

shock. Such plates are candidates for a knock-in event. Again, these plates are usually obvious

in our hands – intensive searching for survivors should not be necessary. Efficiency can vary,

but we routinely recover between two and six independent candidate knock-ins lines from each

set of injections.


• Using a fluorescence dissecting scope, verify that the worms that survived heat shock also lack

expression of the red fluorescent co-injection markers. From each candidate knock-in plate,

single 8–10 non-Unc animals lacking co-injection marker fluorescence to new plates (some of

these worms will be sterile after the heat shock, so don’t try to get away with picking fewer).

• After these worms produce progeny, look at the plates and identify those that did not segregate

Unc progeny. These are likely to be homozygous knock-in strains. Failure to identify any

homozygotes may indicate that your genomic modification is homozygous lethal, or it may be a

sign that you don’t have a true knock-in. Use PCR to distinguish between these two

possibilities. If you are making a GFP knock-in, you can also look for fluorescence at this stage

to establish whether you have an integrant.

PCR verification

• Design primers that anneal inside and outside the homologous repair template (see Fig. 3a for an

example).

• Isolate genomic DNA from the strain to be characterized, using detergent and proteinase K

digestion followed by phenol-chloroform extraction (for a detailed protocol, see

http://thalamus.wustl.edu/nonetlab/ResourcesF/worm%20genomic%20DNA.pdf). A

crowded 5 cm plate yields more than enough DNA. Also isolate DNA from your parental strain

as a control.

• Set up PCR reactions with primer pairs similar to those shown in Fig. 3a–b. We have found that

LongAmp Taq DNA polymerase from NEB gives particularly robust amplification from

genomic DNA.

• Note: the primer pairs outside the homologous repair template (flanking the insertion site) are

particularly useful since you should see a band shift for a knock-in, and the disappearance of the

wild-type band indicates that your strain is homozygous.

• If desired, sequence the PCR products obtained in this step to verify a mutant allele.

Removal of the selectable marker with Cre recombinase

These instructions assume you are using unc-119 selection, but the same basic approach should be

applicable to other selectable markers.

• The night before injection, pick 25–30 L4 knock-in worms to a fresh plate and allow them to

mature into young adults overnight.


• Prepare an injection mix containing the following:

o 50 ng/µL pDD104 (Peft-3::Cre::tbb-2 3’UTR; Addgene #47551)

o 2.5 ng/µL pCFJ90 (Pmyo-2::mCherry pharyngeal co-injection marker) or any other

fluorescent marker.

• Inject the mixture into the gonads of young adult worms. Place the injected worms at 25°C,

three worms per plate.

• Two days after injection, single 10–20 F1 progeny that express the fluorescent marker to new

plates. These animals are progeny resulting from successful injections and can segregate Uncs in

the next generation. Do not put more than one animal per plate at this stage – more animals will

cause the plates to be overcrowded and make screening difficult.

• When the F2 progeny have reached adulthood, pick Unc animals to new plates to establish lines.

Timing is important here: at 25°C, unc-119 animals are slightly egg laying-defective (Egl), and

therefore are easiest to spot when they are old enough to have accumulated some eggs.

However, it is important not to wait too long, as screening becomes more difficult once the

plates become crowded.

• Note: because our Cre expression construct also carries unc-119(+), only animals that have 1)

Excised both genomic copies of the unc-119(+) cassette and 2) Lost the extrachromosomal array

generated by injecting the Cre expression construct will have an Unc phenotype. Loss of the

array can be verified by loss of the fluorescent co-injection marker.

• If desired, confirm removal of the selectable marker by PCR or sequencing. At this stage, the

Unc animals resulting from this procedure can be used in another round of homologous

recombination to produce more complicated modifications, or outcrossed to remove the unc-119

mutation.


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