Zinc finger proteins bind DNA through zinc finger motifs. Each motif contains a beta sheet and alpha helix coordinated by a zinc ion. Early research found zinc fingers bind DNA in the major groove, with fingers 2-5 binding DNA directly and fingers 1-2 interacting through protein-protein interactions. Later, zinc finger proteins were engineered to cut DNA at specific sites, with cleavage occurring near the binding site. This led to the founding of Sangamo Biosciences to develop a modular approach to engineering zinc fingers to target desired DNA sequences. Zinc finger nucleases can introduce double-strand breaks to promote genome editing through homology directed repair.

1. Zinc Finger
Technology
(Review)
Made By:
Munish Chhabra
Roll no-2K10/BT/008
IVth Semester

2. Zinc Finger
– Protein motif that forms a compact globular
structure that coordinates one or more Zn
ions.
– Consensus Sequence-
– 2 Cys held in beta sheet and 2 His in alpha
helix tetrahedrally coordinated to Zn ion.
“zinc finger” describe
finger-like appearance
of the diagram of Xenopus laevis
transcription factor IIIA discovered
In 1985.

4. • Results-
• 5 Zn fingers bound to
21 bp of DNA.
• Finger 2-5 fit in major
groove.
• The alpha helix of the
finger with N terminal
closest to base.
• Conserved regions
recognized by finger
4 and 5.
• Finger 1 and 2 held
by protein-protein
interaction via His
linkers.

6. PCR and fusion????

7. • CP-QDR and
Sp1-QNR Zn finger
Proteins were taken.
• Sp1-QNR cleaved
Lambda DNA(48.5 kb)
to 9.5kb and 39kb.
• CP-QDR cleaved into
5.5 kb and 43 kb.
• Kinetics of cleavage
1) Optimum T=22 o C
2)Efficiency decrease with
increase in KCl
concentration.
3)Efficiency increases with
in crease in MgCl2
Concentration but leading to
nonspecific cleavage.

8. • 300bp-Lambda DNA of major
cleavage site isolated.
• Substrate labeled with 32P at
top and bottom .
• Both cleave near binding sit at
top and bottom at two distinct
locations.

9. And Then….
• Sangamo Biosciences was found in
1995 with a vision to engineer Zn
finger proteins to fit desired base
sequence like Lego blocks.
• The sequences were overlapped.
• Built library of two finger proteins.
• Individual fingers of different target
sites mixed and matched in order to
create a protein with a novel target
sequence --“Modular Assembly” (MA).

11. • ZFN creating double
stranded breaks
(DSB) leads to
Homology directed
Repair (HDR).
• ZFN invoked DSB
increases the rate of
homologous
recombination (HR).
• Maximal HR
frequency when DSB
evoked closer to
mutated site.

13. And Now….
• Keith Joung gave a
new technique-
Oligomerized
Pool Engineering
(OPEN).
• Trancriptor activator
like effectors
(TALENs) on
their way!!!!!!!!!

16. T he End