Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 68

B. Genome Engineering by Homologous Recombination in C. elegans
Over the last 20 years, several attempts were made to develop a genome engi-
neering technique using different strategies to promote the recombination between
the genome and a homologous transgene.
First, it was hypothesized that extrachromosomal arrays made by DNA microin-
jection in the germ line syncitium were bad recombination substrates because of
their repetitive structure ( Broverman et al., 1993 ). Therefore alternative ways of
introducing DNA in the germ line were tested. Engineered DNA fragments were
directly injected into the nuclei of meiotic oocytes. Genomic integration of the
injected DNA was identified in the progeny of the injected animals. Out of the
recombination events, 3% (2 out of 63) resulted from homologous recombination
between the injected DNA and the genome. However, this technique was not further
developed, mostly because of the difficulty to inject DNA into meiotic oocyte nuclei.
Similarly, DNA sequences introduced in C. elegans by biolistic were shown to
recombine at low frequency with homologous genomic sequences ( Berezikov
et al., 2004; Jantsch et al., 2004 ). The use of a visible counterselection marker
was later proved to be very useful to identify rare homologous recombination events
among all transformants ( Vazquez-Manrique et al., 2010 ). However, the homolo-
gous recombination frequency obtained with this method is low. About one recom-
bination event was obtained among 300 transformants derived from 30 independent
bombardment experiments. In a laboratory where biolistic transformation is already
established, performing and screening 30 independent bombardment experiments
will take approximately 2 months of full-time bench work and additional efforts will
still be required to identify bona fide recombinants.
A second set of strategies was using endogenous C. elegans DNA transposons of
the Tc family. DNA transposons move by a ''cut-and-paste'' mechanisms: they
encode a transposase that binds the terminal ends of the transposon and catalyzes
the excision and reinsertion of the transposon DNA, leaving at the excision site a
DSB that must be repaired by the cellular machinery. It was demonstrated that such
DSB can sometimes be repaired by homology-dependent recombination between the
broken chromosome and DNA provided in trans as a repair template ( Gloor et al.,
1991; Plasterk, 1991; Robert et al., 2008 ). This provided a means to copy sequence
variations from the repair template into a genomic target region. The feasibility of
such strategy was demonstrated in C. elegans by Plasterk and Groenen ( Plasterk and
Groenen, 1992 ) and later revisited to establish a genome engineering protocol
( Barrett et al., 2004 ). However, the use of endogenous transposons has a number
of disadvantages. Because these elements are not mobile in the germ line of wild-
type animals, transposon mobilization is achieved in genetic backgrounds, known as
mutators, where germ line transposition is derepressed. As a result, transposons
accumulate in the genome of these mutator strains, causing uncontrolled mutations
resulting in a high morbidity of the strains. In addition, the frequency of homologous
recombination events remains modest, necessitating the growth and screening of
large populations of animals in which the insertion of interest is unstable.