Transcription Activator Like Effector Nucleases or TALENs (Part 17- CRISPR in gene editing and beyond)
Welcome to the 17th part of the multi-part series on applications of CRISPR in gene editing and beyond.
The other gene editing technique is Transcription Activator Like Effector Nucleases abbreviated as TALENs. This technique is similar to Zinc Finger Nucleases in some aspects like ZFNs, TALENs too have 2 domains: one is the DNA binding domain and the second is the DNA cleavage domain. DNA cleavage domain in both cases is the non-specific nuclease FokI enzyme to cleave the genome at a specific targeted site. But the major difference in the two techniques is the difference in their DNA binding domains. As already discussed in Part 15, the DNA binding domain in the case of the zinc finger nuclease technique is derived from the zinc finger motifs. On the contrary, the DNA-binding domain of TALEN is derived from transcription activator-like effector proteins or TALEs. These proteins are secreted by plant pathogen Xanthomonas bacteria, and these proteins act as transcription factors to alter the transcription of genes in host plant cells, thus promoting their successful infection.
A TALE protein consists of a tandem array of repeated segments flanked by N and C terminal domains. Each repeated segment consists of about 34 amino acids. The repeated segments adopt a unique two-helix-shaped structure, with the hypervariable amino acid residues at positions 12 and 13 of each repeated segment making base-specific contacts with the DNA in the major groove.
Therefore, the amino acid sequence of all the repeats is mostly the same except for the amino acids at positions 12 and 13.
The variable pair of amino acids at positions 12 and 13 is most likely responsible for the binding of one segment of TALE protein to a specific nucleotide in DNA. For instance
· A pair of histidine (H) at position 12 and aspartate (D) at position 13 can bind to cytosine (C) in DNA.
· A pair of asparagine (N) and glycine (G) can bind to thymine (T)
· A pair of asparagine (N) and isoleucine (I) can bind to Adenine (A) in DNA
· And a pair of two asparagine (N) residues can bind to Guanine in DNA.
In short, each TALE repeat in an array binds to a single base of DNA. On the contrary, in the case of zinc finger nucleases, each zinc finger repeat recognizes a DNA triplet. So a large number of tandem repeated segments are required for the TALE protein to efficiently bind to a particular nucleotide sequence in DNA.
These tandem repeats of TALE protein can be engineered to recognize and bind to the desired target DNA. If you know the nucleotide sequence of the genomic region you wish to alter, then a TALE protein can be synthesized by assembling the sequence of repeats that are able to recognize the desired target DNA. The sequence of amino acids of variable pairs at positions 12 and 13 of all the repeats must be designed so that they can recognize and bind to the nucleotides of the desired DNA because amino acids at positions 12 and 13 are responsible for binding to DNA. The rest sequence of repeats remains the same.
Thus with 17 repeats of TALE protein, you can uniquely target a 17 base pair DNA sequence.
Similar to Zinc Fingers, TALE protein with repeated arrays, by themselves, can just bind to the target DNA but can’t cleave it. So for using it for genome editing purposes, a DNA-cleaving domain is required to be fused to the TALE to form TALEN. The DNA cleavage domain of the TALENs technique is also derived from the endonuclease enzyme FokI, like in the case of zinc finger nucleases.
While understanding Zinc Finger nucleases, we have discussed that the nuclease domain of the FokI enzyme requires dimerization for endonuclease activity on the target DNA. Thus for fulfilling this condition, 2 TALE protein constructs are required, and then a FokI monomer is fused to each of the two TALE protein constructs. This also facilitates the TALE protein to bind to DNA effectively. Once the TALE protein has bound to the target DNA, the dimerized FokI nucleases can cleave the target DNA by introducing a double-stranded break.
The double-stranded break can be repaired either by Non-homologous end joining or by homologous recombination repair mechanisms, that we have already discussed in Part 16.
These repair mechanisms can then be exploited to achieve targeted genome engineering, such as gene knockout, gene addition to insert DNA of interest, or altering the code of a gene. TALENs may also be useful for gene editing in humans for treating genetic disorders like sickle cell anemia or cystic fibrosis.
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