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Wednesday 26 December 2012

transcription activator–like effector nucleases

In 2012, genome engineers got their hands on some potentially powerful new tools that promise to put the modification of DNA within easy reach of biologists studying a variety of organisms, including yeast and humans.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research.

These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain.

ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations.

One of these tools, called TALENs (for “transcription activator–like effector nucleases”), can destroy or alter specific genes in zebrafish, Xenopus toads, and livestock.

A TALEN is a protein that cuts DNA in specific places, and the ensuing repair modifies the target gene. One group of researchers used the technique to create a miniature pig useful for studying heart disease. Others are modifying the genomes of rats, crickets, and even human cells from patients with disease.

Crystal structures of these effector proteins attached to DNA have revealed how the proteins find their targets.

And at least three teams have come up with a way to make many of these proteins fast and cheaply. This progress has prompted more investigators to give this approach a try.

Such a boom in genome engineering was unthinkable just a few years ago. For most higher organisms, changing or deleting DNA has generally been a hit-or-miss proposition.

Researchers could not readily control where an added gene would insert itself into a genome or which DNA they delete in so-called knockout experiments.

As a result, pinpointing what specific genes do and correcting disease genes in people have posed major challenges.

A decade ago, a new technology called zinc finger nucleases provided a way to target specific genes. Researchers leaped to develop this tool. But zinc fingers proved difficult to make, and one company holds all the key patents. So excitement swelled again in 2009, when two teams discovered a one-to-one correspondence between the repetitive regions of transcription activator–like effector proteins and the DNA bases they attach to, thus providing a new way to target genes. In 2012, studies drove home that TALENs work as well as zinc fingers do but are far easier and cheaper to make. Some researchers now think TALENs will become standard procedure for all molecular biology labs.

Meanwhile, another gene-targeting technology is beginning to make a name for itself.

One drawback of zinc finger nucleases, TALENs, and another genome-editing tool called meganucleases is that they must be reengineered for each new DNA target.

These proteins have two parts: the DNA targeting section and the DNA-cutting section.

The new technology substitutes RNA—which is simpler to make than a piece of a protein—for the DNA targeting section.

It also makes use of a bacterial protein called Cas9, which is part of a natural bacterial defense system called CRISPR, to do the cutting.

Researchers have shown in a test-tube that they can combine these two RNAs into a single one that both matches the DNA target and holds Cas9 in place.

Using this system, they were able to cut specific target DNA, demonstrating the potential of Cas9 to work like TALENs.

Now, those researchers are trying this approach in organisms other than bacteria, and other genome engineers are quite excited about their prospects, suggesting that it may one day challenge zinc finger nucleases and TALENs as the core genome engineering technology.

See also

- genome Engineering

Open references

- TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Mussolino C, Alzubi J, Fine EJ, Morbitzer R, Cradick TJ, Lahaye T, Bao G, Cathomen T. Nucleic Acids Res. 2014;42(10):6762-73. doi : 10.1093/nar/gku305 PMID: 24792154 [Free]

- Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, Zhu Z, Lin S, Zhang B. Nucleic Acids Res. 2013 Aug 1;41(14):e141. doi : 10.1093/nar/gkt464 PMID: 23748566


- ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Gaj T, Gersbach CA, Barbas CF 3rd. Trends Biotechnol. 2013 Jul;31(7):397-405. doi : 10.1016/j.tibtech.2013.04.004 PMID: 23664777

- A. N.-S. Mak et al., “The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target,” Science 335, 716 (10 February 2012).

D. Deng et al., “Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors,” Science 335, 720 (10 February 2012).

D. F. Carlson et al., “Efficient TALEN-mediated Gene Knockout in Livestock,” PNAS 109, (23 October 2012).

D. Reyon et al., “FLASH Assembly of TALENs for High-throughput Genome Editing,” Nature Biotechnology30 460 (May 2012).

J. Kaiser, “Putting the Fingers On Gene Repair,” Science 310, 1894 (2005)

M. Jinek et al., “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337 816-21 (17 August 2012).

V. M. Bedell et al., “In vivo Genome Editing Using a High-efficiency TALEN System,” Nature 491, 114 (1 November 2012).

Y. Lei et al., “Efficient Targeted Gene Disruption in Xenopus Embryos Using Engineered Transcription Activator-like Effector Nucleases (TALENs),” PNAS 109 (23 October 2012).