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An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans Graphical Abstract

Authors Katsufumi Dejima, Sayaka Hori, Satoru Iwata, Yuji Suehiro, Sawako Yoshina, Tomoko Motohashi, Shohei Mitani

Correspondence [email protected]

In Brief Balancer chromosomes are critical tools for genetic research. Using the CRISPR/ Cas9 system, Dejima et al. established a collection of balancer chromosomes in C. elegans. The toolkit will be useful not only for maintenance of lethal/sterile mutants but also for several other applications through customization to suit a particular use.

Highlights d

Generation of a set of structurally defined and aneuploidyfree balancer chromosomes

d

Reliable and easy analyses of embryonic and early larval lethal phenotypes

d

Utilization of the balancers for isolating lethal alleles using the CRISPR/Cas9 system

Dejima et al., 2018, Cell Reports 22, 232–241 January 2, 2018 ª 2017 The Author(s). https://doi.org/10.1016/j.celrep.2017.12.024

Cell Reports

Resource An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans Katsufumi Dejima,1,4 Sayaka Hori,1,4 Satoru Iwata,1,3,4 Yuji Suehiro,1 Sawako Yoshina,1 Tomoko Motohashi,1 and Shohei Mitani1,2,5,* 1Department

of Physiology, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan Women’s Medical University Institute for Integrated Medical Sciences, Tokyo, Japan 3Present address: Center for Education in Laboratory Animal Research, Chubu University, Aichi, Japan 4These authors contributed equally 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2017.12.024 2Tokyo

SUMMARY

Balancer chromosomes are critical tools for genetic research. In C. elegans, reciprocal translocations that lead to aneuploidy have been widely used to maintain lethal and sterile mutations in stable stocks. Here, we generated a set of aneuploidy-free and structurally defined crossover suppressors that contain two overlapping inversions using the CRISPR/Cas9 system. The toolkit includes 13 crossover suppressors and covers approximately 63% of all C. elegans coding genes. Together with the classical intrachromosomal crossover suppressors, the system now covers 89% of the coding genes. We also labeled the created balancers with fluorescent and phenotypic markers. We show that the crossover suppressors are better for embryonic analysis compared with translocational balancers. Additionally, we demonstrate an efficient method to generate lethal alleles by targeting essential genes on a chromosome balanced with a crossover suppressor. The toolkit will allow more efficient experiments in which lethal and sterile mutants can be analyzed. INTRODUCTION Phenotypic analysis from knockouts of essential genes in model organisms has provided a profound understanding of gene function. In C. elegans, 10.3% of genes were estimated to be essential for development or reproduction (Kamath et al., 2003). However, knockout animals of a large number of these genes still remain to be isolated and analyzed. For efficient knockout studies of the essential genes, it is crucial to establish resources that help with the maintenance and analysis of lethal or sterile mutants. Genetic balancers, such as inversions and reciprocal translocations, are important tools to maintain deleterious mutations in heterozygotes. Genetic recombination is significantly prevented within the regions covered by these chromosomal rearrange-

ments (Muller, 1918; Sturtevant, 1921). In addition, balancers can be used for region-specific mutagenesis screens (Zetka and Rose, 1992; Go¨nczy et al., 1999; Justice, 1999). Classically, the chromosomal rearrangements have been made by random mutagenesis, including X-ray and g ray treatments (Ashburner et al., 2005; Edgley et al., 2006). The generation of inversions and translocations in human cell lines and mouse somatic cells has been achieved by genome editing methods, including the Cre-LoxP, transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems (Smith et al., 1995; Choi and Meyerson, 2014; Sander and Joung, 2014). Other studies have also established genome editing methods for chromosomal rearrangements in the germlines of model organisms, including mouse, rat, zebrafish, and nematode (Zheng et al., 1999; Blasco et al., 2014; Chen et al., 2015; Li et al., 2015; Iwata et al., 2016). Although the methodology to induce chromosomal rearrangements using genome editing methods has been well developed, there are no large-scale resources for structurally defined artificial balancer chromosomes covering the whole genome in any model organism. In C. elegans, reciprocal chromosomal translocations, simple chromosomal inversions, free duplications, and crossover suppressors made by random mutagenesis have been used as genetic balancers to maintain lethal and sterile mutations in heterozygotes (Herman et al., 1976; Rosenbluth and Baillie, 1981; Dea`k, 1985; Edgley and Riddle, 2001; Zetka and Rose, 1992). The crossover suppressors, each of which is thought to have an inversion with uncharacterized intrachromosomal rearrangements, are more useful than the others, although reciprocal translocations and simple inversions already covered a large part of the C. elegans genome (Edgley et al., 2006). The reciprocal chromosomal translocations lead to a translocationderived aneuploidy effect whereas inversions and crossover suppressors do not. In theory, the crossover suppressors are more stable than simple inversions because they have more complex structures and, thus, have a greater effect in preventing genetic recombination. However, the classical crossover suppressor alleles (qC1, sC1, sC4, and mnC1) cover only approximately 26% of all C. elegans genes or 24% of the C. elegans genome in terms of physical distance. In addition, these classical balancers harbor unknown structures. We

232 Cell Reports 22, 232–241, January 2, 2018 ª 2017 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. Map of the Regions Covered by the Crossover Suppressors Each chromosome is represented by a gray bar, with cross lines indicating the break points. The chromosomal rearrangements we made in this study are indicated by yellow bars, and the classical chromosomal rearrangements are indicated by light green bars. Fluorescent insertions are shown as triangles; Pmyo-2::Venus and Pmyo-2::gfp are filled in green, and Pmyo-2:: mCherry is filled in red. The detailed structures of each crossover suppressor are shown in Figures S1–S3.

recently established a CRISPR/Cas9-mediated chromosomal editing method that induces structurally complicated intrachromosomal rearrangement and created an artificial complex intrachromosomal rearrangement, tmC1, that covered approximately 5% of all C. elegans genes (Iwata et al., 2016). However, the majority of the C. elegans genome was not covered by these intrachromosomal balancers. Here we describe the creation of a set of strains that possess structurally complex but well-defined crossover suppressor balancers with various markers. We show that embryonic phenotypic analysis is easy and reliable using these balancers. We also describe a methodology to create lethal mutants using the CRISPR/Cas9 system in the presence of the balancer chromosome. The balancer strains we describe will be useful to many C. elegans researchers and will contribute to future study of essential gene function. RESULTS Generation of Crossover Suppressors Using the CRISPR/Cas9 System We aimed to create a set of crossover suppressors that cover a large part of the C. elegans genome in addition to the regions the classical crossover suppressors qC1, mnC1, sC1, and sC4 already covered (Edgley et al., 2006). To this end, we created 13 crossover suppressors covering approximately 89% of all C. elegans genes (18,074 of 20,320 coding genes), or 85% of the C. elegans genome in terms of physical distance, together with the classical crossover suppressors (Figure 1; Figures S1–S3). Overall, we planned to make the crossover suppressors by applying two sequential and overlapping inversions, where the secondary inversion inverts the region between two positions located inside and outside of the primary inversion, as we had constructed tmC1 previously (Figure 2; Iwata et al., 2016).

To introduce the primary inversions, we designed two single guide RNAs (sgRNAs) in the exons of two target genes that were not expected to result in lethal or sterile phenotypes when they were disrupted. This strategy allowed us to obtain chromosomal rearrangements in homozygotes. We used single-stranded oligonucleotides (ssODNs) that joined the chromosomal break points together, each of which had 33 bp of sequence corresponding to each predicted junction point, so that chromosomal rearrangements could be induced by homologous recombination between the targeted regions and ssODNs. The lig-4(tm750) mutant background was used as the recipient for the chromosomal engineering because it showed higher genome editing efficiency than wild-type animals (Iwata et al., 2016). We first generated simple inversions covering various regions of the genome. Additionally, we obtained an inversion that was found in worms treated with trimethylpsolaren (TMP)/UV (Gengyo-Ando and Mitani, 2000; Y.S., unpublished data). The obtained simple inversions are summarized in Table S1. Most of these inversions do not have any visible markers, with a few exceptions (tmIn5, tmIn19, and tmIn26). Next we introduced the secondary inversions on the primary inversions as we had done previously (Iwata et al., 2016). The methods for the secondary inversions were essentially the same as for the primary inversions. The introduction of the secondary inversion not only makes the covered region of the balancer chromosomes larger than simple inversions alone but also more strongly prevents the effects of genetic rearrangements because of increased structural complexity (Figure 2). We obtained 13 crossover suppressors that were largely viable in homozygotes, although tmC29 showed a semi-Emb phenotype for unknown reasons. A balanced heterozygous strain, unc-64(e246)/tmC29, was used for further genome engineering, and the others were used in homozygotes. Seven crossover suppressors (tmC20, tmC18, tmC27, tmC6, tmC25, tmC16, and tmC24), had neither visible recessive nor dominant phenotypes, which indicates that these should be better for further genome engineering. Five crossover suppressors (tmC5, tmC9, tmC3, tmC12, and tmC30), derived from tmIn5, tmIn19, and tmIn26, had visible recessive phenotypes associated with the break points of the primary inversions. However, it was possible to directly inject plasmids into the germline of these homozygous

Cell Reports 22, 232–241, January 2, 2018 233

Figure 2. An Example Schematic of the Crossover Suppressor Construction (A) The Crossover suppressor was created by multiple inversions. Genome regions are colored to show the rearrangement. (B) PCR amplification of break point junctions in wild-type (WT) and tmC24 animals. (C) Break point sequence alignments of ssODN and tmIn60 (top, first inversion) and of ssODN and tmC24 (bottom, second inversion) rearrangements. The tmC24 right break point contained an 11-bp insertion.

animals. The generated crossover suppressor alleles are summarized in Table 1. To label the balancer chromosomes, we added transgenic insertions, which induced Venus or GFP and/or mCherry expression under control of the pharynx-specific myo-2 promoter, to the genes for which loss of function was expected to cause a visible recessive phenotype with decreased reproductive fitness, such as the dumpy (Dpy) and uncoordinated movement (Unc) phenotypes. In the case where the transgenic insertion did not cause any strong phenotype, we further added a mutation to another gene in which loss of function led to a visible recessive phenotype. Furthermore, we obtained crossover suppressors that had only recessive phenotypes but no fluorescent markers for experiments in which fluorescence interferes with the following analyses. For the autosomal balancer chromosomes, the male heterozygotes for the crossover suppressors with recessive mutations mated well. For two types of X chromosome balancers, tmC1 and some tmC24 variants, the hemizygous males showed a recessive Unc phenotype and were not able to mate. We created additional strains in which males carrying

234 Cell Reports 22, 232–241, January 2, 2018

these X chromosome balancers had the ability to mate. Previously, we rescued the Unc phenotype caused by unc-18 disruption in a tmC1 homozygous hermaphrodite with a PCR product of wildtype unc-18 to enable injection of the genome editing plasmids into the gonads (Iwata et al., 2016). We confirmed that tmC1 males carrying the extrachromosomal (Ex) arrays of unc-18(+) can successfully mate. For tmC24 variants that had mutations in the unc-9 gene, we established rescued strains carrying an Ex array of the wild-type unc-9 genome and confirmed that they can mate. The other X chromosome balancer, tmC30, had a break point in the lon-2 gene, and tmC30 hemizygotes exhibiting the long (Lon) phenotype can mate well. The lig-4 mutation was crossed out and was absent in the final strains. The crossover suppressor strains with a green fluorescent marker were further analyzed by whole-genome sequencing, as we have performed previously (Iwata et al., 2016). We confirmed that all of them had only the introduced structural rearrangements. We also found that there was no fatal variation in the analyzed crossover suppressors, suggesting that all genes in the covered region would be stably balanced. Table 2 provides a summary of the strains included in the balancer toolkit in this study. Phenotypic Analysis of Embryos from Animals Balanced with a Created Crossover Suppressor Theoretically, crossover suppressors are much more tractable than reciprocal translocations because they do not lead to aneuploidy (Edgley et al., 2006). To compare the usefulness of the crossover suppressor and the translocation balancer in embryonic analysis, we examined embryonic phenotypes of rab-5 deletion mutants wherein depletion by RNAi was shown to cause variable defects during embryogenesis (Audhya et al., 2007; Hyenne et al., 2012; Patel and Soto, 2013). Although embryonic epidermal fusion defects in a rab-5 deletion mutant ok2605 were analyzed previously (Smurova and

Table 1. List of the Crossover Suppressors Genotype

Left Gene

Right Gene

Covering (Mb)

Chromosome I tmC20: In(F53G12.8 T02E1.7 In(gsp-3 sre-23))

F53G12.8

sre-23

8.1 (0.1..8.3)

tmC18: In(B0207.10 dnj-27 In(gsp-3 sre-23))

gsp-3

snj-27

7.2 (4.7..11.9)

tmC27: In(ile-1 Y18D10A.2 In(dnj-27 dkf-1))

ile-1

dkf-1

ZK1240.1

asm-1

4.6 (2.3..6.9)

hlh-4

ttr-52

3.7 (9.7..13.4)

4 (9.6..13.6)

Chromosome II tmC6: In(sri-57 asm-1 In(ZK1240.1 F29A7.8)) Chromosome III tmC29: In(T05G5.2 Y39E4.1 In(Y39A1A.23 F11F1.7)) Chromosome IV tmC25: In(mak-2 unc-8 In(kvs-5 dmd-9))

kvs-5

unc-8

6.5 (0.7..7.2)

tmC5: In(C01B10.3 eak-7 In(mec-3 unc-31))

C01B10.3

unc-31

6.2 (6.6..12.8)

tmC9: In(glb-19 lgc-52 In(mec-3 unc-31))

mec-3

lgc-52

4.8 (10.5..15.2)

tmC16: In(flp-34 C04E6.7 In(srbc-66 T10H9.8))

flp-34

T10H9.8

5.6 (1..6.7)

tmC3: In(unc-83 C27A7.1 In(unc-23 lon-3))

unc-83

lon-3

5.7 (6.5..12.2)

tmC12: In(hlh-10 C01G10.10 In(unc-23 lon-3))

unc-23

C01G10.10

6.1 (8.9..15.1)

tmC30: In(Y102A11A6 R09F10.1 In(lon-2 mec-10))

Y102A11A.6

mec-10

6.4 (2..8.5)

tmC24: In(mec-10 Y7A5A.20 In(odr-7 F59F4.2))

mec-10

F59F4.2

7.4 (8.5..15.8)

Chromosome V

Chromosome X

Podbilewicz, 2016), whether the parental contribution of rab-5 supports early embryogenesis and how the rab-5 null mutant embryos develop were still unclear. We used two molecular null alleles for this analysis: tm2456 and tm2471 (Figure 3A). They were balanced with fluorescence-labeled tmC18 or hT2 (for simplicity, in the following sections we refer to these balancers as tmC18g and hT2g, respectively, in which ‘‘g’’ means green-labeled). Then we compared the frequency and superficial phenotypes of the fluorescence-negative animals that were produced from rab-5 mutant mothers balanced with tmC18g or hT2g (Figures 3B–3D). We expected variable embryonic phenotypes in the rab-5 mutants based on the reported phenotypes caused by rab-5 RNAi. However, analysis using the tmC18g balancer revealed that rab-5 null mutants exhibited apparently normal early embryogenesis and arrested at the 3-fold or L1 stage (Figures 3E–J). Although RNAi phenotypes for knockdown of rab-5 are distinct, this is not surprising, given the differences between an RNAi knockdown strategy that targets most maternal and zygotic activity from a genetic strategy that targets zygotic activity fully. The worms balanced with tmC18g produced approximately 25% fluorescence-negative progeny, consistent with a Mendelian inheritance ratio, and those fluorescence-negative progeny developed into the 3-fold or later stages with a very subtle corpse engulfment defect (Figure 3C). Thus, the analysis using tmC18g suggested that the parental contribution of rab-5(+) allowed the knockout animals to develop normally during embryogenesis. In contrast, rab-5 mutants balanced with hT2g laid more fluorescencenegative dying progeny than those with tmC18g, and approximately 60% of fluorescence-negative progeny showed defective embryogenesis before the comma stage, presumably because of aneuploidy caused by a translocated chromosome

without the fluorescent marker (Figures 3F and G). Therefore, it was hard to distinguish heterozygotes and homozygotes based on fluorescence alone. Similarly, heterozygotes the of other translocational balancer, nT1, produced arrested embryos, as those of hT2 did (Table S4). The differences in the frequency of lethal embryos are not due to different brood sizes: tm2456/tmC18g, tm2471/tmC18g, tm2456/hT2g, and tm2471/hT2g hermaphrodites laid 222 ± 22, 293 ± 20, 295 ± 13, and 272 ± 18 (mean ± SEM, n = 45) progeny, respectively. These observations demonstrated that tmC18g was better than hT2g for embryonic analyses and suggested caveats in interpreting the embryonic phenotypes when researchers use translocational balancers, as has been suggested elsewhere (Edgley et al., 2006). Generation of Lethal Alleles on Chromosomes Balanced with a Crossover Suppressor Using CRISPR/Cas9 Recently, CRISPR/Cas9-mediated mutagenesis has been widely used to disrupt genes (Chen et al., 2014; Friedland et al., 2013). However, lethal and sterile genes are often difficult to disrupt because of the lethality caused by disruption of both copies of the target gene in the germline as well as by unwanted edits in somatic cells (Shen et al., 2014). In addition, to avoid losing the obtained lethal and sterile alleles, the isolated alleles have to be balanced with appropriate balancers as soon as possible, and this process is often labor-intensive. To overcome these problems, we designed a methodology wherein one of two copies of the gene was selectively edited using CRISPR/Cas9-mediated mutagenesis in the presence of a non-targeted crossover suppressor balancer in heterozygotes (Figure 4A). We attempted to disrupt a potentially essential gene, C47E12.7, that was expected to have severe


Table 2. List of Strains Included in the Balancer Toolkit Strain Name

Allele Name

Genotype

Is Genotype

Phenotypes

Outcrossed

Comments

FX30126

tmC1 X

tmC1

–

Lon Unc Mec

x3

Iwata et al., 2016

FX19397

tmC1 X; tmEx4487

tmC1; unc-18(+)

–

Lon Mec (Unc)

x0

Iwata et al., 2016 Ex+ (pharynx-Venus) males can mate.

FX30133

tmC3 V

tmC3

–

Lon Unc

x2

FX30134

tmC3[tmIs1228] V

tmC3[egl-9(tmIs1228)]

Pmyo-2::Venus

Lon Unc

x2

FX30135

tmC3[tmIs1230] V


Pmyo-2::mCherry

Lon Unc

x2

FX19666

tmC5 IV

tmC5

–

Mec Unc

x2

FX30140

tmC5[tmIs1220] IV

tmC5[F36H1.3(tmIs1220)]

Pmyo-2::Venus

Mec Unc

FX30143

tmC6 II

tmC6

–

FX30144

tmC6[tm9710] II

tmC6[dpy-2(tm9710)]

–

Dpy

x2

FX19668

tmC6[tmIs1189] II

tmC6[dpy-2(tmIs1189)]

Pmyo-2::Venus

Dpy

x2

FX30138

tmC6[tmIs1208] II


Pmyo-2::mCherry

Dpy

x2

FX30151

tmC9 IV

tmC9

–

Unc Mec

x2

FX30234

tmC9[tmIs1221] IV

tmC9[F36H1.2 (tmIs1221)]

Pmyo-2::Venus

Unc Mec

x2

FX30154

tmC12 V

tmC12

–

Lon Unc

x2

FX30152

tmC12[tmIs1194] V


Pmyo-2::Venus

Lon Unc

x2

FX30153

tmC12[tmIs1197] V


Pmyo-2::mCherry

Lon Unc

FX30162

tmC16 V

tmC16

–

FX30164

tmC16[tm9712] V

tmC16[unc-60(tm9712)]

–

Unc

x2

FX30233

tmC16[tmIs1210] V

tmC16[unc-60(tmIs1210)]

Pmyo-2::Venus

Unc

x2

FX30161

tmC16[tmIs1237] V


Pmyo-2::mCherry

Unc

FX30166

tmC18 I

tmC18

–

FX30238

tmC18[tm9705] I

tmC18[dpy-5(tm9705)]

–

Dpy

x2

FX30167

tmC18[tmIs1200] I


Pmyo-2::Venus

Dpy

x2

FX30168

tmC18[tmIs1236] I


Pmyo-2::mCherry

Dpy

FX30176

tmC20 I

tmC20

–

x2

FX30177

tmC20[tmIs1219] I


Pmyo-2::Venus

x2

FX30235

tmC20[tm9709] I

tmC20[dpy-5(tm9709)]

–

Dpy

x2

FX30179

tmC20[tmIs1219 tm9715] I

tmC20[unc-14(tmIs1219) dpy-5(tm9715)]

Pmyo-2::Venus

Dpy

x2

FX30185

tmC24 X

tmC24

–

Mec

x2

FX30240

tmC24[tmIs1240] X

tmC24[F23D12.4(tmIs1240)]

Pmyo-2::Venus

Mec

x2

FX30123

tmC24[tmIs1233] X

tmC24[F23D12.4(tmIs1233)]

Pmyo-2::mCherry

Mec

x2

FX30237

tmC24[tm9723] X

tmC24[unc-9(tm9723)]

–

Unc, Mec

x2

FX30194

tmC24[tmIs1240 tm9719] X

tmC24[F23D12.4(tmIs1240) unc-9(tm9719)]

Pmyo-2::Venus

Unc, Mec

x2

FX30252

tmC24 [tmIs1240 tm9719] X; tmEx4950

tmC24 [F23D12.4(tmIs1240) unc-9(tm9719)]; unc-9(+)

Pmyo-2::Venus

Mec, (Unc)

x2

FX30186

tmC24[tmIs1233 tm9718] X

tmC24[F23D12.4(tmIs1233) unc-9(tm9718)]

Pmyo-2::mCherry

Unc, Mec

x2

FX30253

tmC24 [tmIs1233 tm9718] X; tmEx4950

tmC24[F23D12.4(tmIs1233) unc-9(tm9718)]; unc-9(+)

Pmyo-2::mCherry

Mec, (Unc)

x2

Ex+ (intestinal-GFP) males can mate.

FX30197

tmC25 IV

tmC25

–

x2

One of the break point is in unc-8, but the Unc phenotype is not detectable.

x2 x2

x2 x2

x2 x2

x2 tmIs1219 is inserted in unc-14, but Unc phenotype is not detectable.

Ex+ (intestinal-GFP) males can mate.

(Continued on next page)


Table 2.

Continued

Strain Name

Allele Name

Genotype

Is Genotype

Phenotypes

Outcrossed

FX30257

tmC25[tm9708] IV

FX30203

tmC25[tmIs1241] IV

FX30205

tmC25[unc-5(tm9708)]

–

Unc

x2


Pmyo-2::Venus

Unc

tmC27 I

tmC27

–

FX30258

tmC27[tm9711] I

tmC27[unc-75(tm9711)]

–

Unc

x2

FX30208

tmC27[tmIs1239] I


Pmyo-2::Venus

Unc

x2

FX30227

tmC29 III

tmC29

–

semi-Emb

x3

FX30259

tmC29[tmIs1259] III

tm750 tmC29[unc49(tmIs1259)]

Pmyo-2::GFP

Unc, semi-Emb

x2

FX30229

tmC30 X

tmC30

–

Lon Mec

x2

FX30218

tmC30[tmIs1247] X

tmC30[ubc-17(tmIs1247)]

Pmyo-2::Venus

Lon Mec

x2

FX30236

tmC30[tmIs1243] X

tmC30[ubc-17(tmIs1243)]

Pmyo-2::mCherry

Lon Mec

x2

FX17650

tmIn1 IV

lin-1(tm5929)/tmIn1

–

Unc

x1

Iwata et al., 2016

FX19170

tmIn2 IV

lin-1(tm5929)/tmIn2

–

Unc

x1

Iwata et al., 2016

FX19059

tmIn3 IV

Y38F2AR.9(tm1986)/tmIn3

–

Unc

x1

Iwata et al., 2016

FX17788

tmIn4 II

mlt-7(tm1794)/tmIn4

–

Dpy

x1

Iwata et al., 2016

FX19161

tmIn5 IV

dpy-20(tm5940)/tmIn5

–

Unc

x1

FX19134

tmIn10 II

tmIn10

–

x2 x2

x2

FX19472

tmIn11 IV

mca-3(tm6395)/tmIn11

–

FX19163

tmIn14 I

unc-15(tm6329)/tmIn14

–

Unc

FX19181

tmIn19 V

lig-4(tm750); tmIn19

–

Dpy

x1

FX19173

tmIn26 X


–

Lon Unc

x0

FX19171

tmIn52 III

lig-4(tm750) tmIn52

–

Lon Mec

x0

Obtained by TMP/UV

x2 x1

FX19585

tmIn54 V


–

x0

FX19702

tmIn58 I

tmIn58; lig-4(tm750)

–

x0

FX19704

tmIn60 X


–

x0

FX19706

tmIn62 IV


–

x0

FX19992

tmIn65 I

tmIn65; lig-4(tm750)

–

x0

FX30225

tmIs1246 II

lin-42(tmIs1246)

Pmyo-2::Venus

Egl

x2

FX30262

tmIs1245 IV

dpy-9(tm9713) kvs5(tmIs1245)

Pmyo-2::Venus

Dpy

x2

FX30266

tmIs1224 X

egl-17(tmIs1224)

Pmyo-2::Venus

Egl

x2

FX30269

tmIs1234 X

egl-17(tmIs1234)

Pmyo-2::mCherry

Egl

x2

phenotypes based on RNAi phenotypes. To apply this strategy, we first introduced two silent point mutations, generating nascent protospacer adjacent motif (PAM) sequences to the targeted genomic regions (the first and final exons of C47E12.7) using the CRISPR/Cas9 system (Figure 4B). As expected, the created allele (tm7168) was viable and fertile in homozygotes. To induce a large deletion and to completely knock out the gene, we cut two sites using two sgRNAs that selectively target tm7168 variations and inserted the Pmyo-2:: Venus construct into the cut region by homologous recombination. We injected genome editing plasmids into the gonads of adult worms that had either tm7168 or fluorescence-labeled tmC5 (tmC5g) in each chromosome. We selected 409 Ex-positive (injection marker-positive) F1 worms, excluding tmC5g homozygous Unc animals, and found that 320 of them were tm7168/tmC5g and 89 of them were tm7168/tm7168. From the Ex-positive tm7168/tmC5g F1 progeny, 9 strains had the

Comments

Iwata et al., 2016

tm7168 sequence selectively replaced with Pmyo-2::Venus, keeping tmC5g unchanged (Figure 4C). From the Ex-positive tm7168/tm7168 F1 progeny, we found that none showed the desired edits. It should be noted that the ratio of Ex-positive tm7168/tm7168 was much lower than expected (there should be approximately 160 Ex-positive tm7168/tm7168 individuals based on Mendelian inheritance), suggesting that a large fraction of the tm7168/tm7168 animals died in the presence of Ex because of disruption of both copies of the C47E12.7 gene by Cas9 in germline and/or somatic cells. As a control, we injected the same mixture of the genome editing plasmids into the gonads of tm7168/tm7168 adults and found that only one of the 409 F1 Ex-positive progeny showed heritable editing. All obtained knockout homozygotes (tmIs1265tmIs1274) showed the L2 arrest phenotype at 100% penetrance (Figure 4D). Thus, the strategy in which one copy of an essential gene is selectively targeted by CRISPR-mediated gene editing


Figure 3. Phenotypic Comparison of the rab-5 Mutants from tmC18g- and hT2gBalanced Hermaphrodites (A) Schematic illustration of the rab-5 gene and deletion alleles. (B–D) Representative images of 3-fold embryos of tm2456/tmC18g (B), tm2456/tm2456 produced from the tm2456/tmC18g hermaphrodite (C), and a dead embryo from tm2456/hT2g (D). The genotype of the embryo in (D) was not identifiable based on GFP fluorescence. The tm2456/tm2456 embryos showed superficially normal embryogenesis in terms of body formation but arrested at the 3-fold or L1 larval stage with subtle cellular defects, including an unengulfed cell corpse (arrowhead). (E–G) hT2g-balanced worms produce GFP-negative embryos with defective embryogenesis, possibly because of aneuploidy. Data are represented as mean ± SEM (E). The frequencies of GFP-negative worms from the hT2g-balanced animals are significantly higher than those from the tmC18g-balanced animals (*p < 0.05). The hT2gbalanced worms produced both GFP-positive (F, red) and negative (G, red) abnormal embryos (D). There are significant differences in the rates of abnormal embryos between tmC18g-balanced animals and hT2g-balanced animals (p < 0.05) (F and G). The tmC18g-balanced worms produced GFP-negative embryos that arrested at the 3-fold or L1 stage. GFP-positive progeny from the tmC18g-balanced worms were essentially normal.

DISCUSSION

enables researchers to efficiently generate strains carrying the balanced lethal allele. Generation of Fluorescent Marker Alleles on the Pairing Centers We were not able to generate any inversions near the pairing centers (PCs) (Iwata et al., 2016). Instead, we generated transgene integrations (Pmyo-2::Venus and/or mCherry) in lin-42, kvs-5, and egl-17 loci, which are located in or near the PC regions of chromosomes II, IV, and X, respectively. kvs-5 is located at the left break point of tmC25, but neither lin-42 nor egl-17 are covered by any balancers (Figure 1; Table 2). Transgene integrations in lin-42 and egl-17 resulted in a recessive Egl phenotype. For the transgene integration in kvs-5, we further added a mutation in dpy-9 to introduce the recessive Dpy phenotype. Although, in theory, it is assumed that recombination can occur between fluorescence-labeled mutations, by checking the phenotype and fluorescence of the progeny every several generations, these integrations are practically useful to balance recessive lethal genes located near the PCs.


Here, we generated 13 crossover suppressors labeled with Venus, GFP, or mCherry. With whole-genome sequencing, we confirmed that the crossover suppressors labeled with a green fluorescent marker had only the introduced rearrangements and that there was no fatal variation, suggesting that these crossover suppressors can theoretically balance all genes in the covered regions. The toolkit includes not only fluorescence-labeled balancers but also labelfree crossover suppressors and simple inversions, some of which were established in our previous study (Iwata et al., 2016), so that researchers can use various options as needed. Furthermore, we established strains bearing fluorescent marker insertions for balancing lethal and sterile mutations occurring in the PCs. Fluorescent marker insertions in the PC regions are expected to cover a total of 517 genes that are not covered by the crossover suppressors if we assume that genes located within 0.5 Mbp of each insertion/mutation site could be stably balanced. Including this estimation, more than 91% of genes are covered by the toolkit in combination with the classical crossover suppressors, which cover 26% of the total genes. In addition to strain maintenance and phenotypic analysis, the toolkit has great potential. (1) It can be used for introduction of lethal and sterile mutations using the CRISPR/Cas9 system, as shown in Figure 4. In this study, we inserted allele-specific PAM sequences before applying this strategy. One might target

Figure 4. Lethal Gene Knockout by Targeting One Chromosome Balanced by the Crossover Suppressor (A) A schematic illustration of the knockout experiment using the crossover suppressor balancer. In the conventional method (left), gene disruption of essential genes is not easy because both chromosomes are modifiable; hence, most animals die. After a desirable allele is obtained, the isolated lethal allele must be balanced as soon as possible for maintenance. In our method (right), we first established two silent point mutations that generate nascent PAM sequences. Then the isolated viable mutant allele is balanced with a crossover suppressor balancer covering the essential gene of interest. Then the gene is deleted and replaced with Pmyo-2::Venus by injecting a genome editing plasmid, including a Cas9-sgRNA vector (and the repair template, optional). The isolated lethal allele is balanced with the crossover suppresser when it is generated. (B) A schematic illustration of knockout of the C47E12.7 gene. Asterisks indicate the sites that were modified by the first round of CRISPR/Cas9. These modifications induce silent mutations with nascent PAMs. The two introduced PAM sites are designed to be cut by Cas9 and filled by homologous recombination. (C) Summary of experimental efficiencies to generate lethal mutants. (D) A representative image of the C47E12.7 gene knockout animals showing the L2 arrest phenotype.

SNPs of some wild isolates, such as the Hawaiian strain, nucleotide variations found in the Million Mutation Project (Thompson et al., 2013), or small non-deleterious deletions found in the TMP/ UV-mutagenized library (Gengyo-Ando and Mitani, 2000) if they generated allele-specific PAM sequences. (2) The crossover

suppressors are applicable to conventional forward genetics in which lethal and sterile mutants can be isolated, as the classical balancers have been used previously (Zetka and Rose, 1992; Go¨nczy et al., 1999). (3) The toolkit can be used to conduct genetic modifier screenings of lethal and sterile mutants using conventional mutagens, such as ethyl methanesulfonate (EMS), or using genome-wide RNAi clones. Progeny from animals balanced with crossover suppressors do not show aneuploidy, and, therefore, the screening would be more efficient compared with animals balanced with translocational balancers. Additionally, because the crossover suppressors that we created are structurally defined, PCR genotyping of the fluorescent marker on the balancer and at the break points of the balancer chromosome can exclude false positive mutations caused by unexpected chromosomal rearrangements that would make the heterozygous animals appear as if they harbor the genetic modifiers. (4) Because the balancers that we created are intrachromosomal, even when two mutations exist in different chromosomes, double mutants for essential genes can be easily constructed using two balancer chromosomes. Through such strains, one can assess the epistasis of two lethal genes. (5) The fluorescence-labeled intrachromosomal balancers would also be useful for the rough mapping of mutations. Additionally, if a point mutation of interest is located in the region covered by a crossover-suppressor balancer, then creating transheterozygotes for the point mutation and the fluorescently labeled balancer chromosome, followed by selecting fluorescent-negative progeny, can allow easy removal of side mutations existing outside of the region covered by the balancer without genotyping. Because the toolkit includes various types of balancers with defined structures, researchers can customize them as needed. For example, GFP markers that are expressed by early embryonic promoters, such as end-1 and end-3 promoters (McGhee, 2013), can be added to the crossover suppressors to analyze early embryogenesis because fluorescent proteins driven by the myo-2 promoter start expressing at the late embryonic stage. Thus, the toolkit will be useful not only for maintenance of lethal and sterile mutants but also for several other applications through customization to suit a particular use. EXPERIMENTAL PROCEDURES Further details and an outline of the resources used in this work can be found in the Supplemental Experimental Procedures. Plasmid Construction and ssODNs We used two types of Cas9/sgRNA plasmids: the ‘‘single guide Cas9/sgRNA plasmid’’ containing one U6 promoter::sgRNA, which is structurally identical to pDD162 (Dickinson et al., 2013), except for the DNA encoding sgRNA, and the ‘‘multi-guide Cas9/sgRNA plasmid’’ containing two U6 promoter::sgRNAs. The former was used to insert Pmyo-2::fluorescent protein constructs and to introduce deletions, and the latter was used to introduce chromosomal inversions. Screening for the Generation of Balancers Using the CRISPR/Cas9 System We used ssODNs as repair templates for the chromosomal inversion. We injected a genome editing plasmid mixture containing multi-guide Cas9/sgRNA plasmids (90–100 ng/mL), ssODNs (10.9 ng/mL each), an injection marker


plasmid (Pmyo-2::Venus, 30 ng/mL), and pBluescript (40 ng/mL) into the gonads of lig-4(tm750) animals and lig-4(tm750) animals possessing the first inversions. In the lig-4(tm750) background, targeted chromosomal rearrangement is more effective than in wild-type animals (Iwata et al., 2016). We PCR-screened F1s and confirmed that heritable editing occurred by checking both F2 and F3 genotypes as described previously (Iwata et al., 2016). We PCR-screened 48–202 gravid F1 progeny per trial and obtained 1 or 2 heritable chromosomal inversions (efficiencies, 0.9%–4.2%). The break point sequences were checked by direct Sanger sequencing, and we confirmed that the cut sites were connected with or without insertions or deletions (indels). The primers for detection of the inverted break points are listed in Table S2. We confirmed all break points of the crossover suppressors by PCR amplification after two rounds of backcrossing with N2 to remove lig-4 and possible unknown mutations. We also confirmed that all fluorescently labeled crossover suppressors can indeed balance lethal or sterile mutants, and segregation of the phenotypes was maintained through more than 10 generations. Screening for Generation of the Fluorescent Marker Insertion Lines We injected single guide Cas9/sgRNA plasmids with PCR fragments as repair templates. We amplified the Pmyo-2::Venus::unc-54 30 UTR and Pmyo-2:: mCherry::unc-54 30 UTR with PCR primers designed to contain 33/33-nt homology arms. The primers used for the single-guide Cas9/sgRNA plasmids and repair templates are listed in Table S3. We injected 50–60 animals per trial and selected integration candidates that showed moderate and uniform pharynx fluorescence that was obviously different from the fluorescence derived from the Ex array. Then we tested whether the fluorescence expression of the candidates was 100% heritable. To isolate tmC29[tmIs1259], we used a CRISPR protocol (Dickinson et al., 2015) in which the Pmyo-2::GFP::myo-2 30 UTR sequence and the self-excision cassette (SEC) of pDD317 (Pmyo-2:: GFP + SEC) were inserted in an exon of unc-49, and then the SEC was excised. The primers used to create the repair templates are shown in Table S3. We confirmed that each marker construct was inserted at the expected site by PCR genotyping before and after backcrossing with N2. Isolation of tm7168 To isolate C47E12.7(tm7168), we used a CRISPR protocol (Dickinson et al., 2013). To create the targeting vector, we cloned a 2,604-bp DNA fragment of C47E12 (11,38713,990) that was amplified by PCR into pPD95.75 and introduced 7 silent point mutations: c12073t (the number indicates the position in C47E12), c12076t, g12115a, t12220c, a12464 g, t13251c, and a13278t. Two point mutations, t12220c and t13251c, resulted in nascent PAM sequences, whereas a12464 g disrupted the EcoRI recognition sequence for genotyping. Two point mutations, c12076t and a13278t, disrupted the DNA sequences corresponding to sgRNAs for CRISPR knockin. We injected the genome-editing plasmid mix, including the targeting vector (120 ng/mL), two single guide Cas9-sgRNA vectors (20 ng/mL each), and Pmyo-3::gfp (40 ng/mL) as an injection marker, into the germline of N2 worms. Then tm7169 was isolated from Ex-positive F1 candidates by checking the a12464t edit that disrupted the EcoRI site. The introduced point mutations were confirmed by direct Sanger sequencing. Primers used for the construction of single guide Cas9-sgRNA vectors and targeting vectors are listed in Table S3. Knockout of C47E12.7 We created two single-guide Cas9-sgRNA vectors that target nascent PAM sequences generated by the t12220c and t13251c mutations. To create the targeting vector, we amplified the Pmyo-2::Venus::unc-54 30 UTR and homologous arms by PCR and cloned the fragments into pPD95.75 using In-fusion (Clontech Laboratories, Mountainview, CA). The injection was done as described above except that we injected the DNA mixture into tm7168/ tmC5g and tm7168/tm7168 animals. Whole-Genome Sequencing Whole-genome sequencing and detection of structural variations were performed as described previously (Iwata et al., 2016). The whole-genome sequencing data reported in this paper have been deposited into the NCBI Sequence Read Archive (SRA).


Statistical Methods Statistical analyses were performed using GraphPad Prism7 software (GraphPad; https://www.graphpad.com/). Pairwise comparisons within multiple groups were carried out via ANOVA followed by Tukey’s post hoc tests. All histogram data were obtained from three or more independent experiments and are presented as the mean ± SEM. Asterisks denote the statistical significance compared with the control: *p < 0.05. DATA AND SOFTWARE AVAILABILITY The accession number for the whole-genome sequencing data reported in this paper is SRA: SRP125775. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, three figures, and four tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2017.12.024. ACKNOWLEDGMENTS unc-64(e246) was provided by the C. elegans Genetics Center, which is supported by the NIH National Center for Research Resources. This work was supported by Japan Agency for Medical Research and Development (AMED) under grant number JP16km0210074. We thank the Mitani lab members for their support. AUTHOR CONTRIBUTIONS Conceptualization, K.D., S.H., S.I., and S.M.; Methodology, K.D., S.H., S.I., Y.S., S.Y., and S.M.; Investigation, K.D., S.H., S.I., Y.S., S.Y., and T.M.; Writing – Original Draft, K.D., S.H., S.I., and S.M.; Writing – Review & Editing, K.D., S.H., S.I., Y.S., S.Y., T.M., and S.M.; Funding Acquisition, S.M. DECLARATION OF INTERESTS The authors declare no competing interests. Received: September 28, 2017 Revised: November 30, 2017 Accepted: December 6, 2017 Published: January 2, 2018 REFERENCES Ashburner, M., Golic, K.G., and Hawley, R.S. (2005). Drosophila: A laboratory handbook Cold Spring Harbor (NY: Cold Spring Harbor Laboratory Press). Audhya, A., Desai, A., and Oegema, K. (2007). A role for Rab5 in structuring the endoplasmic reticulum. J. Cell Biol. 178, 43–56. Blasco, R.B., Karaca, E., Ambrogio, C., Cheong, T.C., Karayol, E., Minero, V.G., Voena, C., and Chiarle, R. (2014). Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 9, 1219–1227. Chen, X., Xu, F., Zhu, C., Ji, J., Zhou, X., Feng, X., and Guang, S. (2014). Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans. Sci. Rep. 4, 7581. Chen, X., Li, M., Feng, X., and Guang, S. (2015). Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans. Genetics 201, 1295–1306. Choi, P.S., and Meyerson, M. (2014). Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5, 3728. Dea`k, A.F.P. (1985). The isolation and genetic analysis of a Caenorhabditis elegans translocation (szT1) strain bearing an X-chromosome balancer. J. Genet. 64, 143–157.

Dickinson, D.J., Ward, J.D., Reiner, D.J., and Goldstein, B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 1028–1034. Dickinson, D.J., Pani, A.M., Heppert, J.K., Higgins, C.D., and Goldstein, B. (2015). Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics 200, 1035–1049. Edgley, M.L., and Riddle, D.L. (2001). LG II balancer chromosomes in Caenorhabditis elegans: mT1(II;III) and the mIn1 set of dominantly and recessively marked inversions. Mol. Genet. Genomics 266, 385–395. Edgley, M.L., Baillie, D.L., Riddle, D.L., and Rose, A.M. (2006). Genetic balancers. WormBook, 1–32. Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Colaia´covo, M.P., Church, G.M., and Calarco, J.A. (2013). Heritable genome editing in C. elegans via a CRISPRCas9 system. Nat. Methods 10, 741–743. Gengyo-Ando, K., and Mitani, S. (2000). Characterization of mutations induced by ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans. Biochem. Biophys. Res. Commun. 269, 64–69. Go¨nczy, P., Schnabel, H., Kaletta, T., Amores, A.D., Hyman, T., and Schnabel, R. (1999). Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis. J. Cell Biol. 144, 927–946. Herman, R.K., Albertson, D.G., and Brenner, S. (1976). Chromosome rearrangements in Caenorhabditis elegans. Genetics 83, 91–105. Hyenne, V., Tremblay-Boudreault, T., Velmurugan, R., Grant, B.D., Loerke, D., and Labbe´, J.C. (2012). RAB-5 controls the cortical organization and dynamics of PAR proteins to maintain C. elegans early embryonic polarity. PLoS ONE 7, e35286. Iwata, S., Yoshina, S., Suehiro, Y., Hori, S., and Mitani, S. (2016). Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9. Sci. Rep. 6, 33840. Justice, M.J. (1999). In Mutagenesis of the mouse germline in Mouse Genetics and Transgenics: A Practical Approach, I.J. Jackson and C.A. Abbott, eds. (Oxford: Oxford University Press). Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237.

Li, Y., Park, A.I., Mou, H., Colpan, C., Bizhanova, A., Akama-Garren, E., Joshi, N., Hendrickson, E.A., Feldser, D., Yin, H., et al. (2015). A versatile reporter system for CRISPR-mediated chromosomal rearrangements. Genome Biol. 16, 111. McGhee, J.D. (2013). The Caenorhabditis elegans intestine. Wiley Interdiscip. Rev. Dev. Biol. 2, 347–367. Muller, H.J. (1918). Genetic Variability, Twin Hybrids and Constant Hybrids, in a Case of Balanced Lethal Factors. Genetics 3, 422–499. Patel, F.B., and Soto, M.C. (2013). WAVE/SCAR promotes endocytosis and early endosome morphology in polarized C. elegans epithelia. Dev. Biol. 377, 319–332. Rosenbluth, R.E., and Baillie, D.L. (1981). The genetic analysis of a reciprocal translocation, eT1(III; V), in Caenorhabditis elegans. Genetics 99, 415–428. Sander, J.D., and Joung, J.K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355. Shen, Z., Zhang, X., Chai, Y., Zhu, Z., Yi, P., Feng, G., Li, W., and Ou, G. (2014). Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. Dev. Cell 30, 625–636. Smith, A.J., De Sousa, M.A., Kwabi-Addo, B., Heppell-Parton, A., Impey, H., and Rabbitts, P. (1995). A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nat. Genet. 9, 376–385. Smurova, K., and Podbilewicz, B. (2016). RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion. Cell Rep. 14, 1517–1527. Sturtevant, A.H. (1921). A Case of Rearrangement of Genes in Drosophila. Proc. Natl. Acad. Sci. USA 7, 235–237. Thompson, O., Edgley, M., Strasbourger, P., Flibotte, S., Ewing, B., Adair, R., Au, V., Chaudhry, I., Fernando, L., Hutter, H., et al. (2013). The million mutation project: a new approach to genetics in Caenorhabditis elegans. Genome Res. 23, 1749–1762. Zetka, M.C., and Rose, A.M. (1992). The meiotic behavior of an inversion in Caenorhabditis elegans. Genetics 131, 321–332. Zheng, B., Sage, M., Cai, W.W., Thompson, D.M., Tavsanli, B.C., Cheah, Y.C., and Bradley, A. (1999). Engineering a mouse balancer chromosome. Nat. Genet. 22, 375–378.


Cell Reports, Volume 22

Supplemental Information

An Aneuploidy-Free and Structurally Deﬁned Balancer Chromosome Toolkit for Caenorhabditis elegans Katsufumi Dejima, Sayaka Hori, Satoru Iwata, Yuji Suehiro, Sawako Yoshina, Tomoko Motohashi, and Shohei Mitani

1

Supplemental Information

2 3

An aneuploidy-free and structurally defined balancer chromosome toolkit for Caenorhabditis

4

elegans

5 6

Katsufumi Dejima, Sayaka Hori, Satoru Iwata, Yuji Suehiro, Sawako Yoshina, Tomoko Motohashi and

7

Shohei Mitani

1

8

Supplemental Experimental Procedures

9 10

Strains

11

Caenorhabditis elegans wild-type strain Bristol N2 was used in this study. Nematodes were grown using

12

standard methods (Brenner, 1974).

13 14

Plasmid construction and ssODN

15

To clone the 19 bp of DNA corresponding to the sgRNA in the single-guide Cas9/sgRNA plasmid, we

16

performed inverse PCR with pDD162 as the template and two primers: one of which had 17 bp at the 3’

17

end of the DNA corresponding to the sgRNA in the 5’ end, and the other had the reverse-complementary

18

sequence of 17 bp at the 5’ end, resulting in 15 bp homologous overhangs. The PCR products were

19

circularized using In-fusion (Clontech). To create multi-guide Cas9/sgRNA plasmids, we used two

20

strategies: restriction enzyme-based cloning and PCR/In-fusion-based cloning. For the restriction

21

enzyme-based cloning, two single-guide Cas9/sgRNA plasmids were created as described above. Then,

22

EcoRI/PstI restriction sites downstream of the U6 terminator in one of the single-guide Cas9/sgRNA

23

plasmids were added by inverse PCR with primers containing EcoRI/PstI restriction sites and circularized

24

by the In-fusion reaction. The insert, U6 promoter::sgRNA::U6 3’UTR, was amplified from the other

25

single-guide Cas9/sgRNA plasmid with a primer pair containing EcoRI and PstI restriction enzyme sites

26

in their 5’ end. Alternatively, to create PCR templates for the insert of the multi-guide Cas9/sgRNA

27

plasmid, we performed inverse PCR with pDD162 as the template and two primers: one of which had 17

28

bp at the 3’ end of the DNA corresponding to the second sgRNA at the 5’ end, and the other had reverse

29

complementary sequence of 17 bp at the 5’ end of the DNA corresponding to the first sgRNA at the 5’ end.

30

We also amplified an insert having “first sgRNA::3’ UTR::second U6 promoter::sgRNA” with a

31

multi-guide Cas9/sgRNA plasmid as the template and two primers, of which one had 17 bp at the 3’ end

32

of the DNA corresponding to the first sgRNA at the 5’ end, and the other had a reverse complementary

33

sequence of 17 bp at the 5’ end of the DNA corresponding to the second sgRNA at the 5’ end. The

34

targeted sgRNA+PAM sequences for chromosomal inversions are listed in Table S2, and the primers used

35

for the cloning of single-guide Cas9/sgRNA plasmid are shown in Table S3. We designed ssODN to join

36

two DNA sequences so that the junction is the center of the predicted cleavage sites which are located

37

within 3 bp of the PAM sequences.

38 39

DNA preparation for injection

40

Plasmids were prepared using Qiagen’s Mini Plasmid Purification Kit (QIAGEN, Hilden, Germany) or

2

41

PureLink HQ Mini Plasmid kit (Invitrogen,. Carlsbad, CA). The repair templates were amplified by PCR

42

and purified using Illustra GFX PCR DNA and a Gel Band Purification Kit (GE Healthcare, Little

43

Chalfont, UK).

44 45

Construction of unc-9 rescue strain

46

Two ng/μl of unc-9 genomic DNA, 50 ng/μl of Pvha-6::gfp, and 148 ng/μl of DNA ladder were

47

injected into the gonads of N2 worms. An established Ex strain (tmEx4950) was mated with the strains

48

carrying tmC24 variants with unc-9 mutation.

49 50

Resource Table

51

Bacterial and virus strains, experimental models: organisms/strains, and recombinant DNA were listed in

52

Resource Table.

53 54

Supplemental figure titles and legends

55 56

Figure S1. Structures of the crossover suppressors (Chromosomes I and II), related to Figure 1.

57

The physical positions of breakpoints in crossover suppressors on I and II. Fluorescent insertions are

58

shown in triangles; Pmyo-2::Venus and Pmyo-2::gfp are in green, and Pmyo-2::mCherry are in red.

59 60

Figure S2. Structures of the crossover suppressors (Chromosomes III and IV), related to Figure 1.

61

The physical positions of breakpoints in crossover suppressors on III and IV. Fluorescent insertions are

62


63 64

Figure S3. Structures of the crossover suppressors (Chromosomes V and X), related to Figure 1.

65

The physical positions of breakpoints in crossover suppressors on V and X. Fluorescent insertions are

66


67 68

Reference

69

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.

70

3

71

Resource Table REAGENT or RESOURCE

SOURCE

IDENTIFIER

OP50-1

Chalfie lab

N/A

wild-type, Bristol isolate

CGC

N2

unc-64(e246) III

CGC

CB246

lig-4(tm750) III

NBRP

FX750

V)

NBRP

FX18578

tm2471 I/hT2 (I;III)

NBRP

FX14525

tm2456 I/hT2 (I;III)

NBRP

FX14522

Bacterial and Virus Strains

Experimental Models: Organisms/Strains

nuo-5(tm2751) V/nT1[qIs51] (IV;

Recombinant DNA CRISPR Plasmid for tmC20

This study

F53G12.8+T02E1.7

Plasmid for tmC18

This study

CRISPR B0207.10+dnj-27 pSI20_CRISPR

Plasmid for tmC27

This study

ile-1+F39B2.5

Plasmid for tmC6

This study

CRISPR sri-57+asm-1 pSI32_CRISPR

Plasmid for tmC29

This study

hlh-4+Y39E4A.1 pSI31_CRISPR

Plasmid for tmC25

This study

mak-2+unc-8

Plasmid for tmC5

This study

CRISPR C01B10.3+eak-7 pSI2_CRISPR

Plasmid for tmC9

This study

glb-19+lgc-52 pSI6_CRISPR

Plasmid for tmC16

This study

flp-34+C04E6.7

Plasmid for tmC3

This study

CRISPR unc-83+C27A7.3

Plasmid for tmC12

This study

pSI3_CRISPR

4

hlh-10+C01G10.10 pSI29_CRISPR Plasmid for tmC30

This study

Y102A11.6+R09F10.1 pSI34_CRISPR

Plasmid for tmC24

This study

mec-10+Y7A5A.20 pSI9_CRISPR

Plasmid for tmIn58

This study

gsp-3+sre-23 pSI40_CRISPR

Plasmid for tmIn65

This study

dnj-27+dkf-1

Plasmid for tmIn52

This study

CRISPR hpr-9+ttr-52 pSI16_CRISPR

Plasmid for tmIn62

This study

kvs-5+lgc-9

Plasmid for tmIn5

This study

CRISPR mec-3+unc-31

Plasmid for tmIn54

This study

CRISPR srbc-66+T10H9.8

Plasmid for tmIn19

This study

CRISPR unc-23+lon-3

Plasmid for tmIn26

This study

CRISPR lon-2+mec-10 pSI10_CRISPR

Plasmid for tmIn60

This study

odr-7+F59F4.2

Plasmid for dpy-2 CRISPR

This study

CRISPR dpy-2

Plasmid for egl-9 CRISPR

This study

pSI27_CRISPR egl-9


This study

CRISPR dpy-5

Plasmid for unc-60 CRISPR

This study

pSI39_CRISPR unc-60


This study

pSI38_CRISPR unc-14

Plasmid for F36H1.3 CRISPR

This study

pSI41_CRISPR F36H1.3

Plasmid for egl-17 CRISPR

This study

CRISPR egl-17

Plasmid for lin-42 CRISPR

This study

CRISPR lin-42_1

Plasmid for F23D12.4 CRISPR

This study

pSI28_CRISPR F23D12.4


This study

pSI47_CRISPR unc-75#1


This study

pSI43_CRISPR unc-5

Plasmid for ubc-17 CRISPR

This study

pSI22_CRISPR ubc-17

Plasmid for kvs-5 CRISPR

This study

CRISPR kvs-5


This study

pKD323

5


This study

CRISPR unc-9 multiG1


This study

pSI47_CRISPR unc-75#1


This study

CRISPR dpy-9 ver.2

Plasmid for unc-49 TV

This study

pKD325

Addgene

#19327

Empty sgRNA)

Addgene

#47549

Pvha-6::gfp

This study

pKD218

Pmyo-2::Venus

This study

pFX_Pmyo-2::Venus

Pmyo-3::GFP

This study

pFX_Pmyo-3::GFP

Plasmid pCFJ90 (Pmyo-2::mCherry) Plasmid pDD162 (Peft-3::Cas9 +

pDD162_C47E12.7_sgRN Plasmid for C47E12.7 CRISPR1

This study

A#3 pDD162_C47E12.7_sgRN

Plasmid for C47E12.7 CRISPR2

72

6

This study

A#4

Figure S1: Related to Figure 1. Structures of the crossover suppressors (Chromosomes I and II). The physical positions of breakpoints in crossover suppressors on I and II. Fluorescent insertions are shown in triangles; Pmyo-2::Venus and Pmyo-2::gfp are in green, and Pmyo-2::mCherry are in red.

Figure S2: Related to Figure 1. Structures of the crossover suppressors (Chromosomes III and IV). The physical positions of breakpoints in crossover suppressors on III and IV. Fluorescent insertions are shown in triangles; Pmyo-2::Venus and Pmyo-2::gfp are in green, and Pmyo-2::mCherry are in red.

Figure S3. Related to Figure 1. Structures of the crossover suppressors (Chromosomes V and X). The physical positions of breakpoints in crossover suppressors on V and X. Fluorescent insertions are shown in triangles; Pmyo-2::Venus and Pmyo-2::gfp are in green, and Pmyo-2::mCherry are in red.