Zinc-finger nucleases or ZFNs (Part 15- CRISPR in gene editing and beyond)
Welcome to the 15th part of the multi-part series on applications of CRISPR in gene editing and beyond.
Zinc-finger nucleases are basically types of artificial restriction endonucleases enzymes. A restriction endonuclease is an enzyme that recognizes a specific sequence of nucleotides within the DNA and then produces a double-stranded cut in the DNA. For instance, natural EcoRI restriction endonuclease recognizes a specific GAATTC sequence within DNA and then cuts at this specific sequence. But the trouble with using natural restriction enzymes for genome editing is that their recognition sequences are really short, between 4–8 base pairs. Therefore, any restriction enzyme can cut in hundreds or thousands of places in the genome. But for genome editing purposes, a restriction enzyme is required that can target a longer desired DNA sequence, ideally long enough for its unique occurrence in the genome. And the Zinc Finger nucleases are one of those enzymes that can cleave the DNA sequence you want to edit.
As the name suggests, Zinc Finger nuclease has zinc finger repeats and a nuclease.
Zinc Finger repeats constitute the DNA-binding domain, and the nuclease constitutes the DNA cleavage domain of the Zinc Finger nuclease. Now let’s first talk about the Zinc finger repeats that constitute the DNA binding domain of Zinc Finger nuclease. Each Zinc Finger repeat is a structural motif of proteins. All proteins are made of basic secondary structure units, either α-helix or β-sheets, determined by hydrogen bonding between the amino acids within a peptide chain. But in a protein, the supersecondary structures formed by the combination of the two or three basic secondary structures, α-helix or β-sheets, are known as “motifs.”
Like in the case of one zinc finger motif, which contains approximately 30 amino acids that fold to form a supersecondary structure containing two antiparallel β sheets and an α helix. In the motif, two Cys and two His residues are coordinated by a zinc atom.
The amino acid residues within the α helix of the motif are responsible to make sequence specific DNA contacts in the major groove of the DNA. Thus by changing one or more of the six critical residues located within or adjacent to the α-helix can alter the DNA-binding specificity of a single zinc finger motif. Each zinc finger motif recognizes and binds to only 3 nucleotides. Therefore for recognition of 9 nucleotides within the DNA target, 3 zinc finger motifs are required. In many species, including humans, proteins containing zinc finger motifs act as transcription factors, i.e., regulate gene expression by binding selectively to gene promoters. An example of a human gene that codes for a zinc finger transcription factor is the EGR1 gene.
For designing ZFN that can target the desired DNA, a polypeptide containing at least 3 Zinc finger motifs is designed such that the motifs are able to recognize and bind to the 9 nucleotides within desired target DNA sequences. But Zinc fingers, by themselves, can just bind to the target DNA but can’t cleave it.
So, if you want to design a protein that can selectively bind a promoter to activate a gene, then only zinc finger motifs are required. But if you want to use zinc fingers to create targeted breaks in DNA, you need a pair of scissors to go with them. Therefore a DNA-cleaving domain is required to be fused to the zinc fingers.
The DNA cleavage domain of ZFN is derived from FokI restriction endonuclease enzyme obtained from the bacteria Flavobacterium okeanokoites. FokI enzyme itself consists of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. The recognition site of the enzyme is 5'-GGATG-3'. Once the enzyme is bound to ds DNA via its DNA-binding domain at the recognition site, the DNA cleavage domain is activated. Activated cleavage domain then cleaves DNA, without further sequence specificity. It cleaves the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site.
This implies the presence of two separate protein domains within FokI: one for sequence-specific recognition of DNA and the other for the non-specific endonuclease activity.
Thus the engineered zinc finger nucleases are formed by fusing the FokI cleavage domain to the Zn finger repeats, which are DNA-binding proteins. Studies have also shown that the nuclease domain of the FokI enzyme requires dimerization for endonuclease activity on the target DNA. Thus a FokI monomer is fused to each of the two Zinc finger proteins. This facilitates the zinc finger nuclease to effectively bind to the 18 bp recognition site within the desired DNA sequence of the target genome. Then dimerized FokI nucleases can cleave the target DNA and produce a double-stranded break.
The double-stranded break can be repaired either by non-homologous end joining or by homologous recombination. And both of these repair processes can be exploited to achieve targeted genome editing, such as gene knockout and gene addition. These pathways have been explained in detail in Part 16.
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