PRESENTED BY
S.R. BHARATHKUMAAR,
I M.Sc BIOTECHNOLOGY 2019-2021 ,
BHARATHIAR UNIVERSITY.

Gene corrections is a technology that gives us the
tools for both repairing and mutating DNA, for
discovering gene functions and for engineering new
genetic variants.

Gene correction technology using tailor made nucleases
such as
Mega nucleases (MNs)
Zinc finger nucleases (ZFNs)
TAL effector nucleases (TALENs)
CRISPR/Cas9

Mega nucleases
 “Molecular DNA scissors” that can be used to replace, eliminate
or modify sequences in a highly targeted way.
 By modifying their recognition sequence through protein
engineering, the targeted sequence can be changed.
Zinc-finger nucleases
 Zinc-finger nucleases are powerful gene modification tools
 The ultimate maneuver in gene therapy is to replace a defective
gene with a normal allele at its natural chromosomal location.
 In principle this has several advantages over providing a
complete therapeutic gene e.g., with a viral vector

TAL Effector Nucleases (TALENs)
Are restriction enzymes that can be engineered to cut specific
sequences of DNA.
They are made by fusing a TAL effector DNA binding domain to
a cleavage domain ( a nuclease which cuts DNA strands).
Transcription activator like effectors (TALEs) can be engineered
to bind to particularly any desired DNA sequence, so when
combined with a nuclease, DNA cut at specific locations
The enzymes introduced into cells for gene correction

CRISPR/Cas9
Clustered Regularly Interspaced Short Palindromic Repeats
Use to understanding, characterizing and controlling DNA; Based
on bacterial immune system; single solution to many problems
CRISPR associated system protein 9; An RNA-guided DNA
endonuclease enzyme associated with CRISPR
Cas 9 protein is responsible for locating and cleaving target DNA,
both in natural and in artificial CRISPR/Cas systems
A prominent tool in the field of genome editing
Advance in investigation, prevention, treatment of diseases, to
increase cropyield and to understand the function of the gene

TARGET DNA
RECOGNITI
ON
DNA
CLEAVAGE
CONSTRUCTION NUCLEASE TARGET
SITE
RECOGNI
TION SIZE
MN Homing
endonuclease
Homing
endonuclease
Randomized mutilation at the
DNA recognition residues and
screening of combinatorial library
22 bp
ZFN Zinc finger
domains
FokI nuclease
domain
Assembly of 3-4 zinc finger
domains and screening for
context dependent binding
specificity
(9 or 12 bp)
9 2
TALE
N
RVD (repeat
variable
diresidue)
repeats
FokI nuclease
domain
Assembly of 8-31 RVD repeats (8–31 bp) 9
2
CRISP
R/Cas9
CRISPR RNA
(crRNA)
or guide RNA
(gRNA)
Cas9 Oligonucleotide synthesis of
gRNA
and molecular cloning
(or RNA synthesis)
20 bp +
“NGG” (*1)
Table 1. Various engineered nucleases

Nucleases Cell type Disease Cause of
disease
Correction
ZFN CD4+ T
cells
AIDS HIV infection Disruption of
CCR5/
ZFN CD34+
HSCs
AIDS HIV infection Disruption of
CCR5/
Disruption CRISPR CD4+ T
cells or
293T cells
AIDS HIV infection Disruption of HIV
LTR or excision
of HIV provius
TALEN iPSCs Hepatitis
C
HCV infection Disruption of
APOB
TALEN Fused
dermal
fibroblasts
Kearns-
Sayre
syndrome
5 kb deletion in
mtDNA
Ellimination of
mutant mtDNA

Nucleases Cell type Disease Cause of
disease
Correction
ZFN Myoblasts Duchenne
muscular
dystrophy
Dystrophin
Dexon 51,
51–53,
51–60
(1 + 3n) bp
frameshift at
exon 50
Frameshift TALEN Immortalize
d myoblasts
Duchenne
muscular
dystrophy
Dystrophin
Dexon 48–
50
(2 + 3n) bp
frameshift at
exon 51
ZFN CD4+ T
cells
X-linked
SCID
X-linked
SCID
1 bp
frameshift at
exon 5

Nucleases Cell type Disease Cause of
disease
Correction
ZFN Immortalize
d B
lymphocytes
X-linked
SCID
IL2Rc
truncation in
or after
exon 5
Knock-in
IL2Rc
cDNA (exon
5–8)
Knock-in MN Immortalize
d myoblasts
Duchenne
muscular
dystrophy
Dystrophin
Dexon 45–
52
Knock-in
exon 45–52
ZFN Mouse liver
containing
hF9
Hemophilia
B
F9 Y155
stop
Knock-in
hF9 cDNA
(exon 2–8)
at intron 1
ZFN iPSCs Down
syndrome
Trisomy 21 Knock-in
Xist to
inactivate
the
trisomic chr
21

Nucleases Cell type Disease Cause of
disease
Correction
ZFN iPSCs Parkinson’s
disease
a-synuclein Thr53Ala (A
> G)
ZFN iPSCs Sickle-cell
anemia
b-globin Val6Glu (T
> A)
Substitutio
n
TALEN iPSCs Metabolic
liver disease
a1-
antitrypsin
Lys342Glu
(A > G)
TALEN iPSCs Epidermolys
is bullosa
COL7A1 Stop613Arg
(T > C)

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