DNA cleavage domain: in order to cleave a DNA, the cleavage domain must dimerize, means, it should have two identical structural subunits. Therefore, two ZFNs are required to target non palindromic DNA site. These two ZFNs monomers bind to DNA target sequence in reverse orientation.
DNA binding domain: DNA binding domain of each ZFN contains 3-4 individual fingers, each capable of recognizing 3-bp long sequence. Thus, a heterodimer ZFN, composed of two 9-bp-long DNA binding domains will recognize an approximately 24-bp target sequence (Weinthal et al 2010)
Applications: ZFNs can be used to induce double stranded breaks (DSBs) in specific DNA sequence. A desired homologous donor DNA segment then could be inserted in this break followed by homologous recombination, which leads to tagged gene replacement. On the other hand, the DSBs in former DNA segment could be joined by non-homologous end joining (NHED), which leads to targeted mutagenesis. Important applications of ZFNs are as follows.
A. Debilitate dominant mutation in heterozygous individuals: ZFNs can be used to incapacitate dominant mutations in heterozygous individuals by making DSBs in the DNA in mutant allele. As discussed earlier, the DSB in DNA could be joined by NHEJ. If this joining happens perfect then there would not be any mutation.
B. Modifications of disease/ disorder causing alleles: ZFNs have positive role to play in modification of disease/ disorder causing alleles specifically the disorders caused by dinucleotide repeat expansion. It’s a kind of mutation where trinucleotide ( e.g. CAG repeat, CGG repeat) repeat in certain genes exceeds the normal e.g. 230 to 4000 CGG repeat in X chromosome cause fragile X syndrome, Huntington’s syndrome is caused by CAC repeat. ZFNs can specifically bind to these repeats and could generated DSBs, which leads to shortening of repeats. These short length trinucleotide repeats could be less dangerous (Mittelman et al 2009).
C. New gene addition: addition of desired gene at desired location in the host genome without causing cell injury or mutagenesis is the success of gene therapy. A designed ZFNs having non-specific cleavage domain of Fok I endonuclease with zinc finger protein could make a site specific DSB and with the help of homologous recombination a gene of interest could be inserted at this position (Kandavelou and Chandrasegaran 2008).
Problems Associated:
Cleavage of non-specific target site: it leads to;
a. Double stranded breaks at several locations which could results in chromosome segment translocations and other structural abnormalities
b. Integration of desired DNA in non-specific site could results in altered protein production which could be toxic / lethal for the individual.
REFERENCES:
1. Kandavelou K; Chandrasegaran S (2008). "Plasmids for Gene Therapy". Plasmids: Current Research and Future Trends. Caister Academic Press
2. Klug A, Rhodes D (1987). "Zinc fingers: a novel protein fold for nucleic acid recognition". Cold Spring Harb. Symp. Quant. Biol. 52: 473â€"82
3. Mittelman, D; Moye, C; Morton, J; Sykoudis, K; Lin, Y; Carroll, D; Wilson, JH (2009-06-16). "Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells". Proceedings of the National Academy of Sciences of the United States of America 106 (24): 9607â€"12
4. Weinthal, D., Tovkach, A., Zeevi, V., & Tzfira, T. (2010). Genome editing in plant cells by zinc finger nucleases. Trends in plant science, 15(6), 308-321.
About Author / Additional Info:
Scientist at Division of Genetics, Indian Agricultural research Institute. Presently doing research on identifying QTLs for genotype x management interaction for resource use efficiency in wheat.