Biology Reference
In-Depth Information
isolated. In principle, zinc-finger domains can be designed to target a broad range
of DNA sequences. Donor DNA can be supplied to allow repair of the break by a
homology-driven process, so that a specific gene can be replaced with a desired
sequence. Alternatively mutations can occur through nonhomologous end joining.
It is possible to introduce specific mutations in the genomes of vertebrates,
plants, and fruit flies with ZFNs. So far, the design of ZFNs is difficult, expen-
sive, and laborious and must be developed for each species (
DeFrancesco 2011,
Isalan 2012
). ZFNs obtained from companies and the Zinc Finger Consortium
cost US$650-$25,000 depending on source and whether they are custom made.
Zinc-finger proteins must fit together in a specific way, so designing and testing
ZFNs can be laborious. Information and other resources for ZFNs are available
through the Zinc Finger Consortium (
http://zincingers.org
)
.
Two genomic loci were modified in the germ line of
D. melanogaster
at a
high frequency, which allowed geneticists to discover gene function (
Beumer
et al. 2006
).
Beumer et al. (2008)
reported high frequencies of gene targeting in
Drosophila
by injecting embryos with ZFNs, resulting in replacement by homolo-
gous recombination in up to 10% of the targets at a given locus. More recently,
Takasu et al. (2010)
produced targeted somatic and germ-line mutations in
Bombyx mori
by injecting ZFNs. Despite these successes, the future use of ZFNs
may be supplanted by a different new technology (TALENs, see Section 9.19.4)
because TALENs may be easier to design and use. However, TALENs are less well
studied, so it is still too early to determine whether TALENs are the better tech-
nology (
DeFranisco 2011, Baker 2012
).
9.19.4 Transcription Activator-Like Effector Nucleases (TALENs)
T
Transcription
A
ctivator-
L
ike
E
ffector
N
ucleases (TALENs) are a synthetic mol-
ecule composed of Transcription Activator-Like Effectors (TALEs) and the
Fok
I
endonuclease. They were discovered in 2007as a tool for modifying genomes
(
DeFrancesco 2011
). TALENs are less expensive and easier to design than ZFNs,
and they can be used to upregulate or to edit genes. TALEs, when fused to the
Fok
I nuclease, cleave DNA when present as a dimer. Thus, TALENs function
in pairs, binding opposing DNA targets across a spacer over which the
Fok
I
domains come together to create a break in the DNA strand.
TAL effectors are a family of virulence factors produced by plant pathogens
(
Xanthomonas
species) that can be imported into nuclei and act as transcrip-
tional activators. When TALEs are injected into a host plant, they bind to specific
host promoter sequences that regulate genes affecting the disease process. The
proteins consist of 17 to 18 repeats of 34 amino acids, arranged side by side. DNA
