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Life Technologies
™
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GeneArt
®
Precision TALs technology overview
Transcription activator–like (TAL) effector proteins are
plant pathogenic bacteria proteins that bind to specific DNA
sequences and act as transcription factors during plant
pathogenesis (Figure 1A). The DNA-binding domains of TALs
have a variable number of amino acid repeats, and within each
repeat, two amino acids dictate a specific nucleotide of the
DNA target. Because of the modular domain structure and
well-defined amino acid-to-nucleotide "code", fusion proteins
containing TALs conjugated with various functional domains
can be targeted to very specific loci within the genome. Using
this approach, GeneArt
®
Precision TALs offer site-specific
delivery of nucleases, activators, repressors, chromatin
modifiers, genomic labels, and cross-linking molecules.
The genome editing processes described here uses pairs of
TALs that are fused to truncated FokI nuclease (Figure 1B).
FokI nuclease functions as a homodimer, and creates a
double-strand break in the DNA flanked by the TAL binding
sites. In the absence of DNA that shares homology across
the region containing the break, the cell's natural machinery
will attempt to repair the break by NHEJ, which can lead to
small insertions or deletions, called indels. In protein-coding
regions, these indels can cause frameshift mutations that
can result in a gene disruption (knockout). When this break is
created in the presence of DNA that shares homology across
the region, HDR can occur that allows the added DNA to be
incorporated at the site of the break. In this manner, specific
bases or sequences can be introduced.
Figure 1. GeneArt
®
Precision TALs technology. (A) TAL effector proteins are pathogenic bacterial proteins that bind to specific DNA sequences and act as transcription factors during plant
pathogenesis. (B) TAL-FokI nuclease fusions function as a homodimer to cleave DNA. A TAL-FokI pair is designed to bind to genomic sequences flanking the target site and to generate
a double-strand break at the desired locus. This break can be repaired by NHEJ, which may result in a gene knockout when an indel is introduced. In the presence of homologous donor
DNA, the break can be repaired by HDR, allowing for directed changes to the genome to be introduced.
A
B
Bacterial cell
Nucleus
TAL effector
Transcripts
DNA binding domain
Functional
domain
Without added homologous DNA:
Non-homologous end-joining
Repairs with indels → Gene disruption
With added homologous DNA:
Homology-directed repair
Insert/replace DNA → Gene edit
FokI
FokI
Methods
