Resource
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
Cell Reports 22, 232–241, January 2, 2018 235
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
tmC3[egl-9(tmIs1230)]
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
tmC6[dpy-2(tmIs1208)]
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
tmC12[egl-9(tmIs1194)]
Pmyo-2::Venus
Lon Unc
x2
FX30153
tmC12[tmIs1197] V
tmC12[egl-9(tmIs1197)]
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
tmC16[unc-60(tmIs1237)]
Pmyo-2::mCherry
Unc
FX30166
tmC18 I
tmC18
–
FX30238
tmC18[tm9705] I
tmC18[dpy-5(tm9705)]
–
Dpy
x2
FX30167
tmC18[tmIs1200] I
tmC18[dpy-5(tmIs1200)]
Pmyo-2::Venus
Dpy
x2
FX30168
tmC18[tmIs1236] I
tmC18[dpy-5(tmIs1236)]
Pmyo-2::mCherry
Dpy
FX30176
tmC20 I
tmC20
–
x2
FX30177
tmC20[tmIs1219] I
tmC20[unc-14(tmIs1219)]
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)
236 Cell Reports 22, 232–241, January 2, 2018
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
tmC25[unc-5(tmIs1241)]
Pmyo-2::Venus
Unc
tmC27 I
tmC27
–
FX30258
tmC27[tm9711] I
tmC27[unc-75(tm9711)]
–
Unc
x2
FX30208
tmC27[tmIs1239] I
tmC27[unc-75(tmIs1239)]
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
lig-4(tm750); tmIn26
–
Lon Unc
x0
FX19171
tmIn52 III
lig-4(tm750) tmIn52
–
Lon Mec
x0
Obtained by TMP/UV
x2 x1
FX19585
tmIn54 V
lig-4(tm750); tmIn54
–
x0
FX19702
tmIn58 I
tmIn58; lig-4(tm750)
–
x0
FX19704
tmIn60 X
lig-4(tm750); tmIn60
–
x0
FX19706
tmIn62 IV
lig-4(tm750); tmIn62
–
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
Cell Reports 22, 232–241, January 2, 2018 237
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.
238 Cell Reports 22, 232–241, January 2, 2018
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
Cell Reports 22, 232–241, January 2, 2018 239
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).
240 Cell Reports 22, 232–241, January 2, 2018
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.
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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
shown in triangles; Pmyo-2::Venus and Pmyo-2::gfp are in green, and Pmyo-2::mCherry are in red.
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
shown in triangles; Pmyo-2::Venus and Pmyo-2::gfp are in green, and Pmyo-2::mCherry are in red.
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
Plasmid for dpy-5 CRISPR
This study
CRISPR dpy-5
Plasmid for unc-60 CRISPR
This study
pSI39_CRISPR unc-60
Plasmid for unc-14 CRISPR
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
Plasmid for unc-75 CRISPR
This study
pSI47_CRISPR unc-75#1
Plasmid for unc-5 CRISPR
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
Plasmid for unc-49 CRISPR
This study
pKD323
5
Plasmid for unc-9 CRISPR
This study
CRISPR unc-9 multiG1
Plasmid for unc-75 CRISPR
This study
pSI47_CRISPR unc-75#1
Plasmid for dpy-9 CRISPR
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.