Zinc Finger motif and Zinc Finger Nuclease.

2. Course Coordinator
Dr. Muhammad Tahir
Professor
Group Members
Zeeshan Khan Awan
Affan Ahmed

3. Contents

4. Basic Concepts
about Zinc
Finger Proteins.
 Zinc Finger
Motif
(characteristics)
1
 History.
2
 Functions of
ZF motif.
3
 Families of
Zinc Proteins
4

5. Zinc Finger Motif
 3D Structural motif of the protein.
 Characterized by the co-ordination of one or
more Zn ions.
 Zinc ion is held in place by two cysteine and
two histidine R group.
 Present in the tandem repeats.
 Interacting with DNA and RNA.
 Zinc ion holding the structure together.
 Each zinc motif consist of the 30 amino acid
and folds into beta beta alpha structure.
 Each finger primarily binds to a triplet
within the DNA substrate.
Characteristics
3-D structure
conserved among
different proteins
that serves a
similar function.
Example
helix-turn-helix
motif
(Durai et al., 2005)

6. Zinc Finger Motifs
Note: The zinc ion, found in 8% of all human proteins, plays an
important role in the organization of their three-dimensional structure.

7. Zinc Finger Motif
These are the most abundant proteins in the
eukaryotes genomes. There functions are diverse
and include;
 DNA recognition.
 RNA packaging.
 Transcriptional activation.
 Regulation of the apoptosis.
 Protein folding and assembly.
 Lipid binding.
Functions
(Laity et al., 2001)

8. Crystal Structure of the Zinc
finger
(Nikola P, Pavietich and Carl O. Pabo)
 Crystal structures of zinc finger-
DNA complexes solved in 1991
and 1993 revealed the canonical
pattern of interactions of zinc
fingers with DNA.

9.  Also known as African
Clawed Toad.
 The word Xenopus means
"strange foot"
and laevis means
"smooth “.
History
 Zinc fingers were first identified in the
Xenopus laevis, in the laboratory of
Aaron Klug.
 Revealed that the binding strength of
the transcription IIIA is because of the
presence of the Zinc coordinating
finger like structures.
(Miller et al., 1985)
 British Chemist and
Biophysicist.
 Winner of 1982 Nobel
prize in Chemistry.
 Develeopment of
crystallographic electron
microscopy and his
structural elucidation of
the important nuclic acid
protein complexes
Sir Aaron Klug

10. Cys2 His2
Treble clef
Fingers
Zinc
Ribbons
Zinc Protein
Families.
(40 types of the zinc
fingers in
UniPortKB)
3
1
2

11. Zinc
Protein
Families.
Cys2 His2
 Largest known DNA binding family
in the multicellular organisms.
 Composed of two Beta layers and
one Alpha helix.
 Two cysteine and two histidine
residues located in certain positions
bind zinc to stabilize the structure.
 Four other amino acid residues
localized in specific positions in the
N-terminal region of the α-helix
participate in DNA binding by
interacting with hydrogen donors
and acceptors exposed in the DNA
major groove
 Plays role in - Development,
differentiation, and suppression of
malignant cell transformation
(Razin et al., 2012)

12. DNA reaper
Mechanism.
Zinc Finger
nuclease Components
of ZFN.
Genome
editing.

13.  (Double Strand Break):
The Endonucleases create specific double-strand breaks (DSBs) at
desired locations in the genome and harness the cell’s endogenous
mechanisms to repair the induced break by natural processes of
homologous recombination (HR) and non-homologous end- joining
(NHEJ).
DNA Repair Mechanism.
 Mechanism present in cell to
repair double strand DNA lesions.
 Only used when there is
homologue piece of DNA present
in the nucleus.
 Assumed to be error free because
of use of template.
 Repair double strand break in
DNA in absence of homologous
template.
 Utilize short DNA sequences
called micro homologies to guide
repair.
 Imprecise repair when overhangs
are not compatible.
Homology Direct Repair Non-Homologous End Joining

14. Genome editing (also called gene editing) is a
group of technologies that give scientists the ability
to change an organism's DNA.
These technologies allow genetic material to be
added, removed, or altered at particular locations in
the genome
Genome Editing

15. Zinc Finger Nuclease
These are the artificial restriction
enzymes.
DNA Binding
Domain
 Most abundant DNA-binding
domains encoded in higher
eukaryotic genomes,
composed of a tandem array
of multiple ZF modules in
which each ZF recognizes a 3
bp DNA subsite.
DNA Cleavage
Domain
FokI typeII restriction
enzyme
Naturally found in
Flavobacterium okeanokoites,
consisting of an N-terminal
DNA-binding domain and a
non-specific DNA cleavage
domain at the C-termainal.
 Cleaving domain of ZFNs
contains type IIs restriction
enzyme FokI.
 ZFNs fuse the cleavage
domain to the C-terminus of
each zinc finger domain.
 Two cleavage domains must
dimerize and cleave DNA.
(Klug, 2010)

16. This portion
Includes…
Applications
Comparison of
ZFN, TALEN and
CRISPR/CAS
Limitations
of ZFN.
Mode of
Action of
ZFN.

17. Working of Zinc Finger Nuclease
 (Double Strand Break):
Two ZFNs with different sequence-
specificities collaborate as a heterodimer to
produce a DSB.

18. AP S/T Q Acid
abi4
CGC-CGC-CGC-CAC-CGTA-GGA-GGA-GGA-GCC
GCG-GCG-GCG-GTG-GCAT-CCT-CCT-CCT-CGG
+471+444
Fokl-RR A A A
TCC Fokl-DD
Z F N _ A A A
Z F N _ T C C
100bp

19.  Functional Genomic/Target Validation
• Creation of gene knockout in multiple cell lines.
• Complete knockout of gene not amenable by RNAi.
 Cell-based Screening
• Creation of knock-in cell line with promotors, fusion tags or reporters integrated into
ungenerous genes.
 Cell Lines Optimization
• Creation of cell lines that produce high yield of proteins or antibodies.
Applications of ZFN.

20. Limitation of Zinc Finger Nucleases
Off target cleavage
Immunogenicity
Non-Modular,
difficult and
laborious
engineering process
Pair of ZFNs
required to target
specific DNA
sequence

21. Comparisons
between ZFN
and other
Techniques.

22. Comparison of ZFN, TALEN and CRISPR/CAS
ZFN TALEN CRISPR/CAS
Zinc Finger Nucleases
Properties
Abbreviation
Transcription activator-like
effector nucleases
Clustered, regularly interspaced,
short palindromic repeat
Off target effect Low HighLow
Time Consumption Long(7-15 days) Long(5-7 days) Short(1-3 days)
Cost High High High
Essential Components Zinc finger proteins + FokI
fusion protein
TALE and FokI fusion protein Guide RNA + Cas9 protein
Design Components Protein Protein DNA
Cleaving strand
Efficiency Poor-33% 33% High-70%
Methylation Sensitivity Sensitive Sensitive No
Double Stranded cleavage Single Stranded cleavageDouble Stranded cleavage

23. Comparison of ZFN, TALEN and CRISPR/CAS
Mode of Action

24. Comparison of ZFN, TALEN and CRISPR/CAS
Mode of Action

25. Comparison of ZFN, TALEN and CRISPR/CAS
Mode of Action

26. ANY QUESTIONS?

27.  Miller J, McLachlan AD, Klug A (June 1985). Repetitive zinc-binding domains in
the protein transcription factor IIIA from Xenopus oocytes. The EMBO Journal.
4(6): 1609-14. PMC 554390. PMID 4040853.
 Durani et al., 2005. Zinc finger nucleases: custom-designed molecular scissors for
genome engineering of plant and mammalian cells. Nucleic Acids Research.
 Rudin et al., 2005. Genetic and Physical Analysis of Double-Strand Break Repair
and Recombination in Saccharomyces cerevisiae. Genetics Society of America.
 Rajat M. Gupta and Kiran Musunuru. Expanding the genetic editing tool kit:
ZFNs, TALENs, and CRISPR-Cas9. The Journal of Clinical Investigation.
References.