Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9.

Genetic balancers (including inversions, translocations and crossover-suppressors) are essential tools to maintain lethal or sterile mutations in heterozygotes. Recombination is suppressed within these chromosomal rearrangements. However, despite efforts to isolate genetic balancers since 19781,2,3,4,5, approximately 15% (map units) of the C. elegans genome has not been covered6 (Supplementary Fig. 1). Because the chromosomal rearrangements generated by gamma-ray and X-ray mutagenesis are random, it is difficult to modify specific chromosomal regions. Here, we used the CRISPR/Cas9 genome editing system to solve this problem. The CRISPR/Cas9 system has enabled genomic engineering of specific DNA sequences and has been successfully applied to the generation of gene knock-outs and knock-ins in humans, rats, mice, zebrafish, flies and nematodes7. Recently, the CRISPR/Cas9 system has been shown to induce inversions and translocations in human cell lines and mouse somatic cells8,9,10. Similarly, inversions up to 57.5 kb have been obtained in the zebrafish germline11. Although a large number of cells can be treated at once for effective CRISPR/Cas9 editing in cell lines, it is more difficult to do so in the germlines of model organisms because of limitations in the ability to introduce genome editing tools. Thus, researchers need an efficient way to engineer the chromosomal structure in multicellular organisms in vivo. In the present study, we established an editing method using the CRISPR/Cas9 system in C. elegans to generate genetic balancers at specific chromosomal sites. The inversions and crossover-suppressors produced were up to 6.7 Mb (~17 cM), lengths 2 orders of magnitude longer than produced in a previous work in the germline of a model organism. To facilitate genomic engineering, we targeted the genome rearrangements in a non-homologous end-joining (NHEJ) mutant background. Our method resulted in a higher proportion of successful rearrangements to generate new balancers. Moreover, we found that the inversion and crossover-suppressor balancers generated in heterozygotes did not result in interchromosomal effects.