1. Volume 22, Issue 1, Pages 232-241 (January 2018)
An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans 
Katsufumi Dejima, Sayaka Hori, Satoru Iwata, Yuji Suehiro, Sawako Yoshina, Tomoko Motohashi, Shohei Mitani 
Cell Reports 
Volume 22, Issue 1, Pages (January 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 232-241DOI: (10.1016/j.celrep.2017.12.024)
Copyright © 2017 The Author(s) Terms and Conditions

3. 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.
Cell Reports  , DOI: ( /j.celrep )
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4. 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.
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5. Figure 3
Phenotypic Comparison of the rab-5 Mutants from tmC18g- and hT2g-Balanced 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 hT2g-balanced 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.
Cell Reports  , DOI: ( /j.celrep )
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6. 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.
Cell Reports  , DOI: ( /j.celrep )
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