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binding is determined by the amino acids at positions 12 and 13 within each
repeat. Boch et al. (2009) and Moscou and Bogdanove (2009) published indepen-
dent papers in which the TALE code was broken so that specific TALEs can be
designed for specific gene sequences ( Deng et al. 2012 ). This technology is pat-
ented and is being developed commercially. Costs for a designer TALEN construct
appears to range from US$5000 to $12,000. This technology has been used to
modify yeast, plants, zebrafish and human cells. Will insects be next?
9.19.5 Meganucleases (or Homing Endonucleases)
Meganucleases , also called homing endonucleases , are sequence-specific endo-
nucleases that are found in a variety of single-celled organisms such as Archaea,
Eubacteria, yeast, algae, and some plant organelles. They are called “mega”
because they recognize sites in the DNA ranging from 12 to 30bp in length
(recall that most endonucleases recognize sequences 4-6bp in length). To date,
600 meganucleases from various unicellular organisms have been identified
and sequenced. Meganucleases can identify a single relevant sequence within a
genome because they have such large recognition sites. The length of their recog-
nition site makes them highly specific and useful tools for genome modification.
Naturally occurring meganucleases function as parasitic elements that use the
double-stranded DNA cell-repair mechanisms to multiply and spread, without
damaging the genetic material of its host. A meganuclease binding to its spe-
cific DNA recognition site induces a double-stranded break at the unique site in
the DNA of a living cell. Once DNA damage is sensed by living cells, DNA-repair
mechanisms are initiated and result in homologous recombination. The specific-
ity of meganucleases gives them a high degree of precision and lower cell toxic-
ity than other naturally occurring restriction enzymes. If a homing endonuclease
inserts on one homologous chromosome, it will induce a double-stranded break
on the normal homolog. Repair of the break, using the chromosome contain-
ing the homing endonuclease as a template, results in the homing endonuclease
being copied on to the wild-type chromosome.
Despite the large number of naturally occurring meganucleases, the prob-
ability of finding a homing site in a chosen gene is low ( Smith et al. 2006,
Fajardo-Sanchez 2008 ). As a result, efforts have been made to design artifi-
cial meganucleases with specificities determined by the researcher. Commercial
sources have modified the recognition site of natural meganucleases in order to
target additional specific genomic DNA sequences. To create tailor-made mega-
nucleases, two approaches were adopted: existing meganucleases were modified
by introducing a small number of variations to the amino-acid sequence and then
selecting the functional proteins on variations of the natural recognition site.
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