Zinc finger nucleases (ZFNs) are engineered restriction enzymes designed to target specific DNA sequences within the genome. Assembly of zinc finger DNAbinding domain to a DNA-cleavage domain.

1. Genes editing by Zinc Finger Nuclease
(ZFN)
‫مربوطه‬ ‫استاد‬
‫نبيونى‬ ‫محمد‬ ‫دكتر‬
‫المحمودى‬ ‫حسين‬: ‫دانشجو‬ ‫اسم‬
‫دكترى‬ ‫مقطع‬

2. Content
 Introduction
 Structurer technical of ZFNs
 Some terms associated with this technique
 Models of repair using this technique
 Therapeutic appliances of Gene Editing
Introduction
Zinc finger nucleases (ZFNs) are engineered restriction enzymes
designed to target specific DNA sequences within the genome.
Assembly of zinc finger DNAbinding domain to a DNA-cleavage
domain.
In this technology, efficient and precise genetic modifications
including “gene disruption”-induction of targeted minor insertions
and deletions-, “gene correction”-discrete base substitution
generated by a homologous donor DNA constructs- and targeted
“gene addition”- transferring the entire transgene into an
endogenous locus
‫ا‬ ‫در‬
‫ی‬
‫ن‬
‫فناور‬
‫ی‬
،
‫تغ‬
‫یی‬
‫رات‬
‫ژنت‬
‫ی‬
‫ک‬
‫ی‬
‫دق‬ ‫و‬ ‫کارآمد‬
‫ی‬
‫ق‬
‫جمله‬ ‫از‬
"‫ژن‬ ‫"اختالل‬
-
‫القا‬
‫ی‬
‫درج‬
‫ها‬
‫و‬
‫حذف‬
‫ها‬
‫ی‬
‫جزئ‬
‫ی‬
‫هدفمند‬
-
"‫ژن‬ ‫"اصالح‬
-
‫جا‬
‫ی‬
‫گز‬
‫ی‬
‫ن‬
‫ی‬
‫پا‬
‫ی‬
‫ه‬
‫گسسته‬
‫ا‬
‫ی‬
‫جاد‬
‫توسط‬ ‫شده‬
‫ساختارها‬
‫ی‬
DNA
‫همولوگ‬ ‫دهنده‬
-
‫و‬
‫ژن‬ ‫"افزودن‬
‫هدفمند‬ "
-
‫ترار‬ ‫کل‬ ‫انتقال‬
‫ی‬
‫خته‬
‫به‬
‫ی‬
‫ک‬
‫درون‬ ‫مکان‬

3. Structure of Zinc Finger Nucleases
Cys2–His2 zinc-finger proteins:
Zinc finger motif or zinc finger domain is a common protein motif
that binds zinc ions and forms a finger like structure. This finger like
structure binds the major groove of the DNA and regulates its
transcription.
ZFNinduced double-strand breaks are subject to cellular DNA repair
processes that lead to both targeted mutagenesis and targeted gene
replacement at remarkably high frequencies (1) in Zinc Finger
Nucleases (ZFNs) DNA cleavage function is mediated by FokI
domain. FokI belongs to type ІІ restriction endonucleases.
‫شکستگ‬
‫ی‬
‫ها‬
‫ی‬
‫رشته‬ ‫دو‬
‫ا‬
‫ی‬
‫ناش‬
‫ی‬
‫از‬
ZFN
‫فرآ‬ ‫معرض‬ ‫در‬
‫ی‬
‫ندها‬
‫ی‬
‫ترم‬
‫ی‬
‫م‬
DNA
‫سلول‬
‫ی‬
‫جهش‬ ‫به‬ ‫منجر‬ ‫که‬ ‫است‬
‫زا‬
‫یی‬
‫جا‬ ‫و‬ ‫هدفمند‬
‫ی‬
‫گز‬
‫ی‬
‫ن‬
‫ی‬
‫فرکانس‬ ‫در‬ ‫هدفمند‬ ‫ژن‬
‫ها‬
‫ی‬
‫بس‬
‫ی‬
‫ار‬
‫باال‬
‫م‬
‫ی‬
‫شود‬
(
1
‫هسته‬ ‫در‬ )
‫ها‬
‫ی‬
‫رو‬ ‫انگشت‬
‫ی‬
(ZFNs)
‫برش‬ ‫عملکرد‬
DNA
‫توسط‬
‫دامنه‬
FokI
‫م‬ ‫انجام‬
‫ی‬
‫شود‬
. FokI
‫اندونوکلئازه‬ ‫به‬ ‫متعلق‬
‫ا‬
‫ی‬
‫نوع‬ ‫محدودکننده‬
ІІ
‫ا‬ .‫است‬
‫ی‬
‫ن‬
‫دامنه‬
‫تنها‬ ‫به‬ ‫ها‬
‫یی‬
‫کاربرد‬
‫ی‬
‫ن‬
‫ی‬
‫ستند‬
‫برا‬ ‫و‬
‫ی‬
‫ن‬ ‫شدن‬ ‫فعال‬
‫ی‬
‫از‬
‫د‬ ‫به‬
‫ی‬
‫مر‬
‫ا‬ .‫دارند‬ ‫شدن‬
‫ی‬
‫ن‬
‫و‬
‫ی‬
‫ژگ‬
‫ی‬
‫کل‬
‫ی‬
‫د‬
‫ی‬
‫ها‬ ‫دامنه‬
‫ی‬
FokI
‫پ‬ ‫ژنوم‬ ‫در‬ ‫هدف‬ ‫خاص‬ ‫برش‬ ‫از‬
‫ی‬
‫چ‬
‫ی‬
‫ده‬
‫پشت‬
‫ی‬
‫بان‬
‫ی‬
‫م‬
‫ی‬
‫کند‬

6. .

9. Some terms associated with this
technique
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes
generated by fusing a zinc finger DNA-binding domain to a DNA-
cleavage domain.
A restriction enzyme, restriction endonuclease, or restrictase is an
enzyme that cleaves DNA into fragments at or near specific
recognition sites within molecules known as restriction sites( ‫سا‬
‫ی‬
‫ت‬
‫محدودیت‬ . To cut DNA, all restriction enzymes make two
incisions((‫ها‬ ‫برش‬, once through each sugar-phosphate backbone of the
DNA double helix (11)

10. 2.DNA-binding domain
The DNA-binding domains of individual ZFNs typically contain
between three and six individual zinc finger repeats and can each
recognize between 9 and 18 basepairs. If the zinc finger domains
perfectly recognize a 3 basepair DNA sequence, they can generate a
3-finger array that can recognize a 9 basepair target site. Other
procedures can utilize either 1-finger or 2-finger modules to generate
zinc-finger arrays with six or more individual zinc fingers.
‫ها‬ ‫دامنه‬
‫ی‬
‫به‬ ‫اتصال‬
DNA
‫تک‬ ‫تک‬
ZFN
‫ب‬ ‫شامل‬ ً‫ال‬‫معمو‬ ‫ها‬
‫ی‬
‫ن‬
‫تکرار‬ ‫شش‬ ‫تا‬ ‫سه‬
‫رو‬ ‫انگشت‬
‫ی‬
‫م‬ ‫کدام‬ ‫هر‬ ‫و‬ ‫هستند‬ ‫جداگانه‬
‫ی‬
‫ب‬ ‫توانند‬
‫ی‬
‫ن‬
9
‫تا‬
18
‫پا‬ ‫جفت‬
‫ی‬
‫ه‬
‫تشخ‬ ‫را‬
‫ی‬
‫ص‬
.‫دهند‬
‫ها‬ ‫دامنه‬ ‫اگر‬
‫ی‬
‫رو‬ ‫انگشت‬
‫ی‬
‫کامل‬ ‫طور‬ ‫به‬
‫ی‬
‫ک‬
‫توال‬
‫ی‬
DNA
‫با‬
3
‫پا‬ ‫جفت‬
‫ی‬
‫ه‬
‫را‬
‫تشخ‬
‫ی‬
‫ص‬
‫م‬ ،‫دهند‬
‫ی‬
‫توانند‬
‫ی‬
‫ک‬
‫آرا‬
‫ی‬
‫ه‬
3
‫انگشت‬
‫ی‬
‫ا‬
‫ی‬
‫م‬ ‫که‬ ‫کنند‬ ‫جاد‬
‫ی‬
‫تواند‬
‫ی‬
‫ک‬
‫سا‬
‫ی‬
‫ت‬
‫هدف‬
9
‫پا‬ ‫جفت‬
‫ی‬
‫ه‬

11. ‫تشخ‬ ‫را‬
‫ی‬
‫ص‬
‫سا‬ .‫دهد‬
‫ی‬
‫ر‬
‫روش‬
‫م‬ ‫ها‬
‫ی‬
‫توانند‬
‫ماژول‬ ‫از‬
‫ها‬
‫ی‬
۱
‫انگشت‬
‫ی‬
‫ی‬
‫ا‬
۲
‫انگشت‬
‫ی‬
‫برا‬
‫ی‬
‫تول‬
‫ی‬
‫د‬
‫آرا‬
‫ی‬
‫ه‬
‫ها‬
‫ی‬
‫رو‬ ‫انگشت‬
‫ی‬
‫شش‬ ‫با‬
‫ی‬
‫ا‬
‫ب‬
‫ی‬
‫شتر‬
‫رو‬ ‫انگشت‬
‫ی‬
.‫کنند‬ ‫استفاده‬

12. Models of repair using this technique

15. Repair outcomes of a genomic doublestrand break, illustrated for the
case of ZFN cleavage. A pair of three-finger ZFNs is shown at the top
in association with a target gene ) open box). If a homologous donor
DNA is provided (solid box, left), repair can proceed by homologous
recombination using the donor as template. The amount of donor
sequence ultimately incorporated will typically decline with distance
from the original break, as illustrated by the shading. Alternatively,
the break can be repaired by nonhomologous end joining, leading to
mutations at the cleavage site. These may be deletions, insertions, and
base substitutions, usually quite localized, but sometimes extending
away from the break.(9)
‫نتا‬
‫ی‬
‫ج‬
‫ترم‬
‫ی‬
‫م‬
‫ی‬
‫ک‬
‫شکستگ‬
‫ی‬
‫ژنوم‬ ‫رشته‬ ‫دو‬
‫ی‬
،
‫نشان‬
‫داده‬
‫برش‬ ‫مورد‬ ‫در‬ ‫شده‬
ZFN
‫شكل‬ ‫در‬
‫هستند‬ ‫زیر‬
.
‫ی‬
‫ک‬
‫جفت‬
ZFN
‫انگشت‬ ‫سه‬
‫ی‬
‫با‬ ‫ارتباط‬ ‫در‬ ‫باال‬ ‫در‬
‫ی‬
‫ک‬
‫داده‬ ‫نشان‬ ‫هدف‬ ‫ژن‬
‫اگر‬ .)‫باز‬ ‫جعبه‬ .‫است‬ ‫شده‬
‫ی‬
‫ک‬
DNA
‫سمت‬ ،‫جامد‬ ‫(جعبه‬ ‫شود‬ ‫ارائه‬ ‫همولوگ‬ ‫دهنده‬
‫ترم‬ ،)‫چپ‬
‫ی‬
‫م‬
‫م‬
‫ی‬
‫نوترک‬ ‫با‬ ‫تواند‬
‫ی‬
‫ب‬
‫دهند‬ ‫از‬ ‫استفاده‬ ‫با‬ ‫همولوگ‬
‫ه‬
‫ادامه‬ ‫الگو‬ ‫عنوان‬ ‫به‬
‫ی‬
‫ابد‬
.
‫سا‬ ‫در‬ ‫که‬ ‫همانطور‬
‫ی‬
‫ه‬
‫توال‬ ‫مقدار‬ ،‫است‬ ‫شده‬ ‫داده‬ ‫نشان‬
‫ی‬
‫اهدا‬
‫یی‬
‫نها‬ ‫در‬ ‫که‬
‫ی‬
‫ت‬
‫م‬ ‫گنجانده‬
‫ی‬
‫اصل‬ ‫شکست‬ ‫از‬ ‫فاصله‬ ‫با‬ ً‫ال‬‫معمو‬ ،‫شود‬
‫ی‬
‫م‬ ‫کاهش‬
‫ی‬
‫ی‬
‫ابد‬
‫د‬ ‫روش‬ .
‫ی‬
،‫گر‬
‫شکستگ‬
‫ی‬
‫م‬ ‫را‬
‫ی‬
‫انتها‬ ‫اتصال‬ ‫با‬ ‫توان‬
‫یی‬
‫غ‬
‫ی‬
‫ر‬
‫ترم‬ ‫همولوگ‬
‫ی‬
‫م‬
‫م‬ ‫برش‬ ‫محل‬ ‫در‬ ‫جهش‬ ‫به‬ ‫منجر‬ ‫که‬ ‫کرد‬
‫ی‬
.‫شود‬
‫ا‬
‫ی‬
‫نها‬
‫جا‬ ‫و‬ ‫درج‬ ،‫حذف‬ ‫است‬ ‫ممکن‬
‫ی‬
‫گز‬
‫ی‬
‫ن‬
‫ی‬
‫پا‬
‫ی‬
‫ه‬
‫موضع‬ ً‫ال‬‫کام‬ ً‫ال‬‫معمو‬ ‫که‬ ‫باشند‬
‫ی‬
‫اما‬ ،‫هستند‬
‫گاه‬
‫ی‬
.‫هستند‬ ‫گسست‬ ‫از‬ ‫دور‬ ‫اوقات‬

16. Therapeutic appliances of Gene Editing
The use of site-specific nucleases for therapeutic purposes represents
a paradigm shift in gene therapy. Unlike conventional methods, which
either temporarily address disease symptoms or randomly integrate
therapeutic factors in the genome, ZFNs and TALENs are capable of
correcting the underlying cause of the disease, therefore permanently
eliminating the symptoms with precise genome modifications. To
date, ZFN-induced HDR has been used to directly correct the disease-
causing mutations associated with Xlinked severe combined immune
deficiency (SCID) specific ‘safe harbor’ locations in the human
genome [4]. Although the overall utility of site-specific nucleases is
currently limited in somatic cells, continued progress in stem cell
research, including the production and manipulation of iPS cells, will
ultimately open countless new directions for gene therapy, including
treatments based on autologous stem cell transplantation. ZFNs have
been used to genetically repair Parkinson’s disease-associated
mutations within the SNCA gene in patient-derived human iPS cells
[5]. Targeted gene knockout via ZFN-induced NHEJ-mediated repair
has also proven a potentially powerful strategy for combating
HIV/AIDs. ZFNs have been used to confer HIV-1 resistance by
disabling the HIV coreceptor C-C chemokine receptor type 5 (CCR5)
in primary T cells [6] and hematopoietic stem/progenitor cells [6].
This approach is currently in clinical trials (NCT01252641,
NCT00842634 and NCT01044654). More recently, ZFNmediated
targeted integration of anti-HIV restriction factors into the CCR5
locus has led to the establishment of T cells that show near-complete
protection from both R5- and X4-tropic strains of HIV [7].
Additionally, ZFNs have been used to improve the performance of T-
cell-based immunotherapies by inactivating the expression of
endogenous T cell receptor genes [8], thereby enabling the generation
of tumor-specific T cells with improved efficacy profiles. Finally,

17. site-specific nucleases afford the unique possibility of safely inserting
therapeutic transgenes into
Reference
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2. McManus, M.T. and Sharp, P.A. (2002) Gene silencing in
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4. Carroll, D. (2011) Genome engineering with zinc-finger nucleases.
Genetics 188, 773–782
5. amirez CL, Foley JE, Wright DA, et al. (May 2008). "Unexpected
failure rates for modular assembly of engineered zinc fingers".
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doi:10.1038/mt.2008.114. PMID 18545224
10.DeKelver, R.C., Choi, V.M., Moehle, E.A., Paschon, D.E.,
Hockemeyer, D., Meijsing, S.H., Sancak, Y., Cui, X., Steine, E.J.
and Miller, J.C., 2010. Functional genomics, proteomics, and
regulatory DNA analysis in isogenic settings using zinc finger
nuclease-driven transgenesis into a safe harbor locus in the human
genome. Genome Research 20, 1133-1142.
11.Doyon, Y., McCammon, J.M., Miller, J.C., Faraji, F., Ngo, C.,
Katibah, G.E., Amora, R., Hocking, T.D., Zhang, L., Rebar, E.J.,
Gregory, P.D., Urnov, F.D. and Amacher, S.L., 2008. Heritable
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12.Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M.H. and
Chandrasegaran, S., 2005. Zinc finger nucleases: custom-designed
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13.Klug, A., 1993. Co-chairman's remarks: protein designs for the
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