This document discusses genome editing tools for genetic engineering, including zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-Cas9. It describes how each works to cut DNA at specific sites, and how this can be used for targeted gene therapy. As an example, CRISPR-Cas9 is highlighted for its potential use in treating genetic diseases like sickle cell anemia through gene editing. Overall, the document outlines current genome editing techniques and their applications, as well as the future potential of CRISPR-Cas9 specifically for cancer treatment and regenerative medicine.

1. M.Sc. Biotech./Biochem./Microbio. (Sem – II)
Genetic Engineering
Gene Manipulation
By – Dr. Ravi Kant
Assistant Professor (Biotechnology)
Email – ravi.kant@nirmauni.ac.in

2. ● INTRODUCTION
● GENOME EDITING TOOLS
❑ Zinc Finger Nuclease
❑ TALENS
❑ CRISPR CAS9
● TARGETED GENETHERAPY
● FUTURE PERSPECTIVE
Components

3. Introduction
Genetic diseases result from aberrant gene expression or loss of its
complete function.
For such genetic diseases, targeted therapies have been introduced to
restore the normal function of a gene.
The targeted gene therapies depend upon the endonucleases that
recognize and cleave at the specific sites and replace the defective
copy with the normal gene. There are classes of rare cutting
endonucleases used for gene targeting: i) naturally occurring homing
endonucleases. ii) engineered chimeric proteins (zinc finger nucleases
and TAL Effector Nucleases. iii) CRISPR/Cas9.

4. GENOME EDITING TOOLS
(1) ZINC FINGER NUCLEASES (ZFN)
(2) TRANSCRIPTION ACTIVATOR LIKE RESTRICTION ENDONUCLEASES(TALEN)
(3) CRISPR / CAS9

5. ZINC FINGER NUCLEASES(ZFN)
• ZFN is an artificial endonuclease
wherein a tailored zinc finger protein
(ZFP) linked to the FokI restriction
enzyme's cleavage domain. By designing
ZFPs with novel sequence specificities, a
ZFN can be modified to cleave new
targets. It is used to cleave a specific
genomic sequence for genome
engineering purposes. The ZFN-induced
cleavage event triggers cellular repair
processes, effectively altering the
targeted locus.
• Flk1, preferred endonuclease, cleaves
DNA sequences non-specifically.

6. TRANSCRIPTION ACTIVATOR LIKE RESTRICTION
ENDONUCLEASE(TALEN)
The FokI nuclease is fused to sequence-specific TAL DNA binding domains to create
TALENs. The transcription activator-like (TAL) modules are derived from plant
pathogenic bacteria and have a normal function of binding particular promoters and
activating transcription in the nucleus of host plant.
Most natural TAL effectors recognize sequences between 15 and 19 bp in length.
Customizing the TAL effector domain has resulted in the creation of targeted
transcription factors.

7. CRISPR/CAS9 ENDONUCLEASE ACTIVITY
CRISPR/Cas9 is a modern molecular biology technique for genome editing systems that
destroys targeted DNA that is present in the cell. In this technique, targeted DNA is cut at a
specific location. After cutting DNA, it may be destroyed or, sometimes, it is repaired by a
repairing mechanism, and that leads to an error-prone mechanism and some sort of
mutation.
CRISPR/Cas9
CRISPR Cas9
(RNA template) protein(nuclease)

8. HOW THE CRISPR/CAS9 CAUSES AN
ENDONUCLEASE ACTIVITY?
CRISPER is a complementary RNA strand for target DNA. After complementary DNA binds with the
targeted DNA, it can be activated. Cas9 has catalytic activity and cuts the targeted DNA.
The first idea of CRISPER Cas9 was to come up with bacterial immunity because bacteria can be
attacked by the phage virus. crRNA works with Cas9 and sometimes it works with trcrRNA (Tresser
RNA) along with Cas9. There are certain types of pathways to work with CRISPER.
1) CrRNa+Cas9
2) CrRNA+trcrRNa+Cas9
Catalytic sites are very special because they have an NGG sequence. N stands for nucleotide, and
this NGG is also known as a PAM site (protospacer adjacent motif). On the protospacer side, where
CrRNA binds with the targeted DNA, In the type 2 mechanism, crRNA and trcrRNA bind with each
other via a loop, and then RNase comes up and cuts the loop, but the role of trcrRNA in the gene
therapy technique is still not known.

9. DIAGRAM OF CRISPR/CAS9 ACTIVITY

10. TARGETING DISEASES THROUGH GENE THERAPY:
HEMATOLOGICAL DISEASE: Individuals affected with Sickle Cell Anemia can be
treated using CRISPR/CAS9 gene editing technology .

11. OTHER DISEASES TREATED BY TARGETED
GENE THERAPY
❑ CARDIOVASCULAR DISEASE e.g. hypertrophic
and Duchenne muscular dystrophy (DMD).
cardiomyopathy (HCM)
❑ CANCER TREATMENT
❑ NEURODEGENERATIVE DISEASES
❑ HEREDITARY EYE DISEASE

12. FUTURE PERSPECTIVE
By using Genome editing technology one can modify the genomic sequence
according to their will . We can generate mutation corrected human induced
pluripotent stem cells for ex vivo regenerative medicine. Most importantly
CRISPR-Cas9 has a bright future ahead in gene editing technology this method
involves cancer treatment by modifying genome of target cells .Adeno
associated viral vector are used for gene editing technology that can be used in
future.So major future targets are crop improvement and treatment of genetic
disorders .