ZINC FINGER NUCLEASES IN GENE EDITING.ppt

CONCEPT AND WORKING MECHANISM

1. ZINC FINGER NUCLEASES
DR. ESTHER SHOBA R
ASSOCIATE PROFESSOR
DEPARTMENT OF LIFE SCIENCES
KRISTU JAYANTI (DEEMED TO-BE UNIVEESITY)

2. INTRODUCTION
•Zinc-finger nucleases are basically types of artificial restriction endonucleases
enzymes.
•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.

3. ZINC FINGER REPEATS
•Zinc Finger repeat is a structural motif of proteins.
•One zinc finger motif, approximately 30 amino acids 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.

5. DNA BINDING REQUIREMENT
•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

6. NUCLEASE OF ZFN
•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.

8. •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.
•Therefore, 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.

13. Non-Homologous End Joining (NHEJ)
•Non-Homologous End Joining is a rapid and efficient double-stranded repair
mechanism that involves a simple ligation of the two DNA ends that result
from a double-stranded break.
•The FokI-mediated DNA cleavage leaves overhanging ends, which can either
be filled in completely or incompletely by the host polymerase enzyme or
chewed back by limited exonuclease activity.
•Then the ligase enzyme ligates the two DNA ends to complete the repair
process.
•As a result of this repair process, small insertions or deletions of nucleotides
can be generated in the region, flanking the ZFN-mediated cleavage site.

15. •When the zinc finger nucleases are targeted to the protein-coding regions,
the outcome is often a shift in the reading frame, usually leading to a null
allele or non-functional allele of the targeted gene. The zinc finger nucleases
combined with repairing by non-homologous end joining can be used for
introducing mutations or for knocking out a gene. Knocking out a gene
means making the gene inactivate or inoperative.

16. EXAMPLE
•In a study, researchers designed a pair of zinc finger nucleases targeted to
the first coding exon of CCR5.
•CCR5 is the major co-receptor for HIV entry and is present on the human T
cell.
•When these ZFNs were introduced into human T cell lines, the zinc finger
nucleases disrupted the CCR5 gene.
•This ultimately resulted in decreased expression of CCR5, as well as
protection from HIV infection because it blocked the entry of HIV into T cells.

17. The first coding exon of the CCR5 gene gets disrupted by ZFN

18. Homology-directed repair (HDR) pathway
•Alternatively, the homology-directed repair pathway, abbreviated as HDR, utilizes the
presence of a template DNA for repairing double-stranded breaks.
•The template DNA carries the desired sequence of the gene of interest and is flanked by
approximately 1 kbp homology on either side of the mutation.
•The template DNA then replaces the target double-stranded locus to restore genetic
information in an error-free manner.

19. •When ZFN binds to the targeted DNA, and the FokI enzyme introduces
double-stranded breaks in it, the introduction of double-stranded breaks
activates the DNA damage repair by homology-directed repair.
•Therefore, after ZFN cleavage, the ends of the target DNA are digested by the
5-3 exonuclease enzyme, leaving 3' overhangs
′ ′
•One of the resulting single-stranded 3 ends invades homologous sequences
′
in the template DNA.
• The invading 3 end is then extended by DNA polymerase represented by a
′
dashed line.
• After some synthesis, the extended end withdraws and anneals to the other
end at the original break.
•The gaps are filled in, and the continuity of the strands is restored by ligation.

21. EXAMPLE
•In a study, the researchers designed ZFNs targeting the IL2Rγ gene, which is
mutated in X-linked severe combined immune deficiency syndrome or SCID.
Specifically, the ZFNs were designed to target exon 5 of the IL2Rγ gene. To
introduce the desired changes, a plasmid containing the unmutated or wild-
type exon with approximately 1 kbp homology on either side of the mutation
was introduced into the cells. Co-transfection of ZFNs and the plasmid
carrying desired DNA into cells replaced the mutated IL2Rγ exon 5 with wild-
type exon 5. Thus, fixing the gene responsible for causing X-linked severe
combined immune deficiency syndrome.

22. SUMMARY
•Zinc Finger Nucleases (ZFNs), a class of engineered endonucleases, facilitate
targeted genome editing by binding to a user-specified genomic locus and
causing a double-strand break (DSB).
•The cell then employs endogenous DNA repair processes, either non-
homologous end joining (NHEJ) or homology-directed repair (HDR), to repair
this targeted DSB.
•These repair processes can be channeled to generate precisely targeted
genomic edits resulting in an organism or cell lines with specific gene
disruptions (knockouts), integrations, or modifications such as disease-
associated SNPs.