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.

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex

“…3C). To quantify the synaptic decrease of the L-AChRs in emc-6 (kr150), we generated a knock-in strain carrying the unc-29 L-AChR subunit endogenously tagged with tagRFP using the MosTIC technique (46). Consistent with immunostaining of LAChRs, we observed an 82% decrease of the amount of L-AChR at the NMJ (Fig.…”

Mos1-mediated insertional mutagenesis in Caenorhabditis elegans

“…Mos1 mutagenesis is safe for the experimenter, in contrast to EMS, which is highly mutagenic in all metazoans, including Homo sapiens. We recently developed a technique called MosTIC to engineer custom C. elegans alleles of genes of interest by homologous recombination 22 . This technique relies on the remobilization of a Mos1 transposon from the targeted locus.…”

Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System

“…In the past few years new methods for genome editing have become available for this model system, all of which involve the creation of a specific double-strand break in the genome. The most widely applied method uses the Mos1 transposon and transposase (Robert and Bessereau 2007;Frokjaer-Jensen et al 2008, 2010. Although this method enables precise genome editing, it is limited by the availability of a transposon at the desired locus.…”