Gene Therapy: The current

solution for many dreadful

diseases.

Soumyadip Das,
Graduate student, Dept. of Biomedical Sc. &
Engineering
Hanyang University, Seoul
Advisor: Dr. Suresh Ramakrishna.

Gene therapy:
Gene therapy is the technology in which any gene,
which is responsible for the development of a disease,
is replaced with a healthy gene.
The gene therapy can be performed with the
aim of gene editing accordingly,
A. Replacing a mutated gene that causes disease with a
healthy copy of the gene.
B.Inactivating, or knocking out, a mutated gene that
is functioning improperly.
C.Introducing a new gene into the body to help to fight
against a disease.

Gene Editing:
The gene editing can be performed with the help
of artificial programmable nucleases or in other
words restriction enzymes, which produce site
specific DNA-double strand break which leads to
mutagenesis.
Types of nucleases:
I.Zinc finger nucleases (ZFNs)
II.Transcription activator-like effector nucleases
(TALENs)
III.RNA-guided engineered nucleases
(RGENs)/CRISPR Cas9 system

Zinc-finger nucleases: (First generation

nucleases)
Artificial restriction enzyme and have two
domains;
DNA binding domain,
genetically engineered
to bind to specific site
of DNA.
It can recognise
between 9-18 bp.
DNA cleaving domain,
Fokl restriction enzyme
used for cleavage of

Transcription-activator like effector

nuclease (TALENs): Second generation
nuclease
Artificial restrition enzyme generated by fusion of,
transcription activator (TAL) proteins or DNA
binding domain
DNA cleaving domain (Fokl nuclease)

CRISPR/Cas9 (cluster regularly interspaced

short palindromic repeat):
Targeting RNA : CRISPR RNA
Trans-CRISPR RNA
Cas9 protein: Which cleaves or break the
double strands
and induce a mutation.

Overlapped Extension
PCR:
1. To Insert specific mutation at
specific points in a sequence.
2.To splice (join) two smaller
DNA fragments into larger
polynucleotide.

Kbp
2000
1st PCR with Primers a+b
1000 After
and c+d,
500
We could see two fragments of
definite sizes.

After 2nd PCR with primers a+d,

We could differentiate and
understand whether the
overlapping has happened or
not, by determining the size of
the band.

1.Transformation.
2.Incubation overnight at 37
degree centigrade.
3.Colonies.
4.Screening those colonies (colony
PCR).
5.Positive colonies are Inoculated
in growth media.
6.Mini-prep of the Positive
colonies.
Send those for sequencing.