Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks

Abstract

The Drosophila element Mos1 is a class II transposon, which moves by a 'cut-and-paste' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool--called MosTIC--to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.

Figure 1

MosTIC strategy and efficiency. (A) Main pathways potentially used to repair a Mos1 excision-induced DSB during MosTIC experiments. In this example, homologous chromosomes (in red and blue) carry the same Mos1 insertion and a homologous repair template is provided on a transgene (in gray). Repair of the broken chromosome (in blue) is based either on end-joining or homologous recombination (single-strand annealing or gene conversion) (see Introduction). (B) Schematic representation of the exon/intron structure of the Mos1-containing alleles unc-63(kr19∷Mos1) and unc-5(ox171∷Mos1) with the repair templates (unc-63.rep, unc-5.repL and unc-5.repS) used in MosTIC experiments. Mos1 elements are indicated by triangles. The restriction sites ApaLI and EcoRV were introduced into the repair templates to identify MosTIC events. The repair templates did not contain enough sequences to rescue the mutant phenotypes. However, copying these sequences into the Mos1-mutated genomic loci would restore functional genes. (C) MosTIC efficiency at the unc-63 and unc-5 loci. Frequencies correspond to the number of phenotypic revertants in the progeny of transgenic animals where Mos1 excision was triggered by heat-shock. MosTIC events were identified among the phenotypic revertants by the presence of an ApaLI site (unc-63 locus) or an EcoRV site (unc-5 locus) copied into their genome. n, number of independent experiments.

Figure 2

Analysis of MosTIC conversion tract. (A) Repair template engineered to monitor the MosTIC conversion tract. The restriction sites and SNPs introduced into the repair template are designated by their position relative to the Mos1 insertion point and are as follows: −4246 nt=AclI, −2816=HindIII, −1983=NheI, −816=Acc65I, −30=SNP1, 0=EcoRV, +120=SNP2, +583=SacI and +1342=SmaI. The conversion tracts analyzed are represented below the repair template scheme. Black and white dots show sites that were present and absent, respectively, in the chromosome after gene conversion. Numbers to the right of conversion tracts indicate the occurrence of each tract (out of 21). (B) The number of occurrences per site was plotted against their position in the repair template. The numbers close to the dots are the raw data. The gray zone corresponds to the 1 kb region centered at the Mos1 insertion point.

Figure 3

Knockout and knock-in by MosTIC at the unc-5 locus. (A) Scheme of the repair templates designed to engineer deletions and insertions in the unc-5 gene. The primers used to screen by PCR for MosTIC-engineered animals are indicated by arrows. Limits of the repair templates are indicated by dotted lines. The EcoRV site present in unc-5.SDgfp is shown. (B) Frequencies of the different events. n, number of independent experiments.

Figure 4

PCR analysis of DSB repair in lig-4/cku-80-defective backgrounds. (A) Details of the experimental procedure used to study DSB repair by PCR. unc-5(ox171∷Mos1) adults were heat-shocked (t=0) to induce Mos1 excision and DSB repair was analyzed by PCR with primers flanking the Mos1 insertion point (t=18 h). PCR products were analyzed on an agarose gel: (−) non-heat-shocked, (+) heat-shocked samples, M=size marker (1 kb Plus DNA Ladder, Invitrogen). (B) Sequence analysis of footprints generated in wild-type and cku-80 and lig-4 mutants. See Table I footnotes for legends. Numbers in parentheses represent the numbers of footprints analyzed per genotype.