TALENs Gene Editing in Plants
Transcription Activator Like Effector Nucleases (TALENs) is one of the three most common techniques that we currently use in gene editing, along with Zinc-Finger Nucleases (ZFNs) and CRISPR-Cas9
By Izabela Ninu
A simplified analogy to understand these methods is to view them as successive iterations of gene editing technology:
ZFNs, being the most basic form, can be thought of as the first version. They laid the groundwork for the development of more advanced techniques. TALENs, which we will delve into, represent the second version. They offer a more refined approach compared to ZFNs but still lack the simplicity, versatility, and precision of the third version, CRISPR-Cas9, which is currently the most widely used due to these advantages.
Origin
TALENs are proteins that originate from Xanthomonas bacteria, a type of plant bacteria. When these bacteria infect plants, they secrete TALENs which contain highly conserved repeats. These repeats, also known as Repeat Variable Di-residues (RVDs), function like a key, enabling specific binding to a particular section of DNA.
TALENs are constructed by fusing the TALEs with Fok1 endonuclease, an enzyme used to cleave DNA at the desired location. In simpler terms, the fusion of TALEs and Fok1 endonuclease allows us to create a highly specific ‘gene scissor’. This ‘scissor’ can then be used to cut the DNA at a precise point, enabling targeted gene editing.
Operation method
Firstly, a TALEN construct, which is specific to the DNA sequences of interest, is engineered. This construct is then inserted into a plasmid (a circular piece of DNA found within a eukaryotic cell)
The plasmid, now carrying the TALEN construct, is transfected into the cell. In other words, the TALEN hitches a ride with the plasmid into the cell. Once inside the cell, the DNA of the plasmid gets expressed, and along with it, the TALEN is also expressed. This allows the TALEN to enter the nucleus of the cell.
Inside the nucleus, the TALEN binds to its specific section of DNA, thanks to the Repeat Variable Di-residues (RVDs). These RVDs function like a key, enabling the TALEN to bind to the DNA at the right location.
The Fok1 endonuclease then creates a cut in the DNA. This process occurs simultaneously on both strands of the double-stranded DNA, resulting in a double-stranded break. By carefully controlling how the TALENs cut the DNA and whether or not this happens in the presence of an alternative DNA strand, a gene can either be deleted or altered.
These are a type of restriction enzyme, which means that they are going to recognize a specific DNA sequence, and they’re going to cut at that specific DNA sequence.
With TALENs, we can create highly specific nucleases. For instance, an adenine nucleotide might have a specific TALEN that binds to it, and thymine might have a different one.
To construct these nucleases, we attach multiple TALENs together to form a large protein. Often, about nine of these TALENs are joined together. Each TALEN recognizes a specific nucleotide — G, C, T, or A. By bonding these TALENs together, we create a protein that doesn’t just bind to one nucleotide, but to a sequence of nine nucleotides. It will only bind if that entire sequence of nine nucleotides is present.
Next, we attach half of an endonuclease enzyme, FOKL, to this protein. FOKL, derived from Flavobacterium, is an endonuclease that becomes active when its two parts combine. We attach the other half of FOKL to a set of TALENs that recognize a complementary strand of DNA. This results in a large protein structure that recognizes a specific sequence of nine nucleotides and is attached to an active FOKL enzyme.
When we introduce these engineered structures into a DNA sample, each structure binds to a specific location. The two halves of the FOKL enzyme then dimerize, forming a functional enzyme that cuts the six nucleotides in between the binding sites. This cut is staggered, leaving overhangs or ‘sticky ends’ of four nucleotides.
What sets FOKL apart is its lack of specificity. Unlike other restriction endonucleases like EcoR1, which require a very specific recognition sequence (G-A-A-T-T-C and its complement C-T-T-A-A-G) to cut, FOKL can work with any combination of nucleotides. This flexibility is why we use FOKL.
The specificity in this process comes from deciding which combination of Transcription Activator-Like Effectors (TALEs) to use in the TALEN construct. The entire structure, consisting of nine TALEs and the two halves of FOKL, is known as a Transcription Activator-Like Effector Nuclease, or TALEN.
TALENs are extremely specific. They require a specific combination of about 18 nucleotides in specific positions. You can make these even longer for more specificity. This is a significant advantage over natural restriction endonucleases like EcoR1, which, when introduced into the human genome, can cut at multiple places due to the prevalence of its recognition sequence. Each TALE recognizes a single nucleotide, offering more flexibility. This means you can build TALENs to recognize any sequence you want, making them a powerful tool in gene editing.
