Abstract
Balancer chromosomes are convenient tools used to maintain lethal mutations in heterozygotes. We established a method for engineering new balancers in C. elegans by using the CRISPR/Cas9 system in a non-homologous end-joining mutant. Our studies will make it easier for researchers to maintain lethal mutations and should provide a path for the development of a system that generates rearrangements at specific sites of interest to model and analyse the mechanisms of action of genes.
Highlights
Background genotypeWT (N2)lig-4 WT (N2)lig-4 lig-4 lig-4 lig-4Confirmation of the suppression of recombination in tmIn3(IV)
We constructed two targeting vectors that joined the chromosomal breakpoints together, each of which had 2 kb of sequence homologous to each predicted junction point, so that chromosomal rearrangements could be induced by homologous recombination (HR) between the targeted regions and homology vectors (Fig. 1b, Supplementary Fig. 2)
A previous study has reported that disabling non-homologous end-joining (NHEJ) via the RNAi inactivation of the cku-80 gene, which acts as a DNA binding protein, significantly improves the HR efficiency in C. elegans[12]
Summary
Confirmation of the suppression of recombination in tmIn3(IV). We examined whether tmIn3(IV) could balance a recessive lethal mutation within the inversion interval, as described in Supplementary Fig. 5a. Heterozygous tmIn3(IV) hermaphrodites were mated with heterozygous males carrying a recessive lethal lin-1 mutation (tm5929). After the self-fertilization of F1 worms, the balanced strain lin-1/tmIn3(IV) segregated three phenotypes: WT (lin-1/tmIn3 heterozygotes), lethal (lin-1 homozygotes) and larval arrest (tmIn3 homozygotes) (Supplementary Fig. 5b–g). The new balancer is a useful tool for maintaining lethal mutations on the left arm of chromosome IV. The segregation of these phenotypes was maintained through more than 20 generations, suggesting that tmIn3 suppresses further recombination of the covered genomic region
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