Two years ago, a new class of proteins exclusive to the Xanthomonas genus of bacteria was discovered [1]. Two groups led by Jens Boch at the Martin-Luther-University, and Halle-Wittenberg and Adam Bogdanove from Iowa State University each independantly published the nucleotide recognition code of the TAL effectors. When added to cells, TAL effectors can be used to edit genomes in situ. TALENs, aka transcription activator-like effector nucleases, are artificial restriction enzymes generated by fusing two TAL effector DNA binding domains to a DNA cleavage domain. TAL effectors have been utilized to create site-specific gene-editing tools by fusing target sequence-specific TAL effectors to nucleases (TALENs), transcription factors (TALE-TFs) and other functional domains.They work much like ZFNs do to create the intended result, be it to knock in genes or knock out mutations. But unlike ZFNs, which are able to bind to groups of three base pairs, TALENs bind only to individual nucleotides, allowing their use anywhere on the genome (see figures 1 and 2).
Two years ago, a new class of proteins exclusive to the Xanthomonas genus of bacteria was discovered [1]. Two groups led by Jens Boch at the Martin-Luther-University, and Halle-Wittenberg and Adam Bogdanove from Iowa State University each independantly published the nucleotide recognition code of the TAL effectors. When added to cells, TAL effectors can be used to edit genomes in situ. TALENs, aka transcription activator-like effector nucleases, are artificial restriction enzymes generated by fusing two TAL effector DNA binding domains to a DNA cleavage domain. TAL effectors have been utilized to create site-specific gene-editing tools by fusing target sequence-specific TAL effectors to nucleases (TALENs), transcription factors (TALE-TFs) and other functional domains.They work much like ZFNs do to create the intended result, be it to knock in genes or knock out mutations. But unlike ZFNs, which are able to bind to groups of three base pairs, TALENs bind only to individual nucleotides, allowing their use anywhere on the genome (see figures 1 and 2).
The DNA binding domain of TALENs contains a highly conserved repeat sequence of 33-34 amino acids in length with the exception of the 12th and 13th amino acid. These two adjacent amino acids are highly variable (termed repeat-variable-diresidue [RVD]) and confer a strong specificity for one of the four DNA base pairs [2-6].
This 1:1 ratio between the amino acids, and the target DNA sequence is what has allowed for the engineering of TALENs, which are basically highly specific DNA binding domains paired with a repeat combination of amino acids and the appropriate RVD. The beautiful and elegant part is that TALENs are completely modular and they pretty much spell out an unambiguous code for the targeted DNA sequence (see below).
Figure 1. Illustration of TALEN design
Image courtesy of: GeneCopoeia. www.genecopoeia.com
Figure 2. Illustration of TALE-EF design
Image courtesy of: GeneCopoeia. www.genecopoeia.com
The Process for Synthesisizing TALENs
Now, to make sense of it all, let's say you have 17 repeats of 34 amino acids, that's 17*34 = 578 amino acids required, plus the non-repeating C- and N-terminal sequences of a TAL effector, and you would be able to uniquely target a 17 base pair DNA sequence. If you used two TALs, you can recognize 34 bases; attach the FokI obligate heterodimer in between them, and you've got a TALEN.
In theory, there are a number of possible TALEN pairs available for each base pair on any random DNA sequence [6].
The RVD code is what genetic engineers use to create many TALE repeats that bind with high affinity to the desired genomic DNA sequences, at rates reported as high as 96% [7, 8, 9].
Perhaps one of the most appealing features of TALENs is the ease with which they can be made. Each one can be designed and assembled in as little as two days and in as large a number as hundreds at a time [10, 11]. Indeed, a library with TALENs targeting all of the genes in the human genome has already been constructed, found
HERE [12].
Here's the graph you've been waiting for showing the beautiful and elegant parts:
| Amino Acids 12 & 13 |
Corresponding Base Pair |
| NI |
A |
| HD |
C |
| NN |
G or A |
| NK |
G |
| NG |
T
|
In effect, this new tool is modernizing the field of genetics so rapidly and in such a way that, experts say, promises easier and safer access to gene-editing technology. Cellectis believes its TALEN technology will greatly speed up the process. We jumped into this technology initially because we could see that it provides a very efficient and rapid means to perform gene editing, cited Philippe Duchateau, Ph.D., CSO in a recent interview with
Genetic Engineering News.
Dr. Duchateau also indicated that, Although TALENs have exquisite programming specificity, one problem is their sensitivity to methylation, a ubiquitous DNA modification. We overcame this major bottleneck by using a combination of biochemical, structural, and cellular approaches and have developed an efficient and universal method to overcome this limitation. Moreover, by coupling end-processing enzymes with nucleases, we improved even further targeted gene disruption in a variety of cell types.
For a 20-page protocol on how to make your own TALENs, see
Sanjana 2012, and for some open source resources to help you design it, see
Cornell University's TALE-NT tools. Or you can buy a custom-designed TALEN from Cellectis, which holds the exclusive commercial license on TALENs, for $5,000/pair.
TALENs are still quite new at this point, but they have already been used in some groundbreaking applications.
Ding 2013 reported on using them to make isogenic models of human disease pairs of cell lines that are (theoretically) identical except for the single site of a disease-causing genetic mutation. Their study found certain cell lines didn't turn out exactly identical: the TALENs did exhibit a low but measureable rate of mutagenesis at off-target sites. But then again, simply growing cells in culture leads to the gradual accumulation of point mutations over time (just as aging leads to gradual accumulation of somatic mutations in cells in your body). So in practice, it seems only a certain small percentage of cells (Ding 2013 reports anywhere from 1.6% to 34%) will take up the new genetic alterations, which you can then fraction out for the rest of your experiments.
This is one reason why these technologies are still a ways away from allowing us to perform gene therapy in every cell of an adult human. But in the immediate future, these are amazing tools for research, allowing us to create new models of human diseases. In the slightly longer term, there is also the possibility that they'll play a role in making it possible to transplant modified versions of your own cells back into your body, similar to an organ transplant. While some might say that's a harder load to haul, one company,
Stem Cells Inc has already brought
human neural stem cell transplants to the clinical development stage, so don't rule it out.
SOURCES
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Science. 2011 Sep 30;333(6051):1843-6.
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[3] Boch J, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009; 326(5959):1509-1512.
[4] Morbitzer R, Romer P, Boch J, Lahaye T. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc Natl Acad Sci US A. 2010; 107(50):21617-21622.
[5] Streubel J, Blucher C, Landgraf A, Boch J. TAL effector RVD specificities and efficiencies. Nat Biotechnol. 2012; 30(7):593-595.
[6] Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol. 2012; 30(5):460-465.
[7] Cong L, Zhou R, Kuo YC, Cunniff M, Zhang F. Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun. 2012; 3:968.
[8] Miller JC, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011; 29(2):143-148.
[9] Li T, et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 2011; 39(1):359-372.
[10] Hockemeyer D, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011; 29(8):731-734.
[11] Cermak T, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011; 39(12):e82.
[12] Kim Y, et al. A library of TAL effec