This presentation provides an overview of genome editing. It defines genome editing as a technique used to modify DNA within a cell precisely and efficiently using engineered nucleases. The presentation discusses different tools for genome editing including meganucleases, zinc finger nucleases, TALENs, and CRISPR. It explains how these tools work by creating cuts in DNA at specific sequences, and how cells naturally repair this using homologous directed repair or non-homologous end joining. Applications of genome editing discussed include gene therapy, modifying crops and animals, disease treatment, and ecological control.

2. PRESENTATION ON Genome Editing
PRESENTed BY:-
Muhammad Aquib
Nasir Husain
M.Sc.(Previous)
Department of Zoology,
AMU, Aligarh

3. GENOME EDITING
INTRODUCTION
GENOME EDITING
BASIC ASPECTS OF GENOME EDITING
TOOLS OF GENOME EDITING
HOW IT WORKS ?
APPLICATIONS
CONCLUSION
REFERENCES

4. INTRODUCTION
• Genome editing, or Genome editing with engineered
nucleases (GEEN) is a type of genetic engineering in which
DNA is inserted, replaced or removed from a genome using
artificially engineered nucleases or molecular scissors.
• These nucleases are specific in nature which create the
double strand breaks (DSBs) at desired location and harness
the cells endogenous mechanism to repair the induce break
by natural processes of homologous recombination (HR) and
non- homologous end-joining (NHEJ).

5. What is genome editing ?
 Genome editing is a technique used to modify DNA within a cell,
precisely and efficiently.
 It involves making cuts at specific DNA sequences with enzymes
called engineered nucleases.
 Genome editing can be used to add, remove or alter DNA in the
genome.
 By editing the genome, the characteristics of a cell or an
organism can be changed.

6. Why genome editing ?
To understand the function of a gene or a protein, one
interferes with it in sequence-specific way and monitors its
effects on the organism.
In some organisms, it is difficult or impossible to perform
site-specific mutagenesis, and therefore more indirect
methods must be used such as silencing the gene of interest
by short RNA interference (siRNA).
Nucleases such as CRISPR can cut any targeted position in
the genome and introduce a modification of the endogenous
sequences for genes that are impossible to specifically target
using conventional RNA interference (RNAi).
To increase the nutritional content and pest resistance in the
crop with knock-out therapies.

7. Basic aspects of Genome editing

8. Tools of Genome editing

9. Tools of Genome editing
Meganuclease-based Engineering:-
• Meganucleases are characterized by their capacity to recognize and
cut large DNA sequences (from 12 to 40 base pairs)
• Best known meganuclease protein is LAGLIDADG family
• Natural meganucleases have certain variation in recognition site.
Zinc finger nuclease based engineering:-
• It occur in several transcription factors.
• It play important role in stabilization of DNA interaction site by
binding towards it.
• The C- terminal of each finger nuclease responsible for the specific
recognition of the DNA sequence.
• The recognized sequences are short made up of around 3 base
pairs.
• It is possible to control the expression of a specific gene.

10.  Transcription activator-like effector nuclease(TALEN)
• It is a artificial restriction enzymes generated by fusing a
specific DNA binding domain to a Non-specific DNA cleaving
domain.
• It is designed to bind at any desired DNA sequence, comes from
TAL effectors.
• TAL effectors consist of repeated domains, each which contains
of highly considered sequence of 34 amino acids, and recognize
a single DNA nucleotide
• In TALENs the binding specificity is higher and off-target
effects are lower.
• The construction of DNA binding domains is easier.

11.  Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR):-
• It play key role in a bacterial defense system.
• It form the basis of a genome editing technology known as
CRISPR-Cas9 that allows permanent modification of organisms.
• CRISPRs are found in approximately 40% of sequenced bacterial
genomes and 90% of sequenced archea.
• It contains a section of genetic code containing short repetitions of
base sequences followed by spacer DNA segments.
• It is a revolutionary technique that can modify any region of the
genome of any species with high precision and accuracy without
harming other genes.

12. How does Genome editing work ?
• Genome editing uses a type of enzyme called an engineered nuclease
which cuts the genome in a specific place.
• Engineered nucleases are made up of two parts:
(a) A nuclease part the cuts the DNA.
(b) A DNA targeting part that is designed to guide the nuclease to
specific sequence of DNA.
• Nuclease acts in following steps:
1) Gene disruption
2) Gene insertion
3) Gene correction
4) Chromosomal rearrangement

13. Nuclease Activity

14. • The disruption process comprises of cutting of DNA in a
specific place and the cell will naturally repair the cut.
• The insertion process is done to induce the nuclease on
the genomic sequence.
• The correction method is mediated by DSB-induced
HR, which can be exploited for gene replacement,
especially for treatment of monogenic diseases.
• Chromosomal rearrangement include large-scale
deletions, insertions, duplications and inversions, which
are associated with many genetic diseases and cancer.

15. PRINCIPLES OF GENOME EDITING
The editing of genome can be done by using:-
o Homologous directed repair (HDR)
o Non Homologous end joining (NHEJ)
Homologous Directed Repair
 The HDR requires longer sequence similarity than NHEJ, which requires
alignments of only a few complementary bases for the ligation of the two
ends.
 HDR is more efficient if the DSB and modification site are within 10
nucleotides.
 To increase the level of repair by HDR, the repair template can be
engineered to have additional homologous sequence both upstream and
downstream of the targeted area.
 HDR mostly occur in S and G2 phases of cell cycle.

16. Working of HDR

17. Non Homologous End Joining (NHEJ)
 NHEJ is more efficient than HDR, but has much lower fidelity and,
therefore, is much error prone, resulting in insertions or deletions.
 NHEJ requires less sequence homology than HDR, does not require
template, entails less DNA synthesis and is faster.
 NHEJ occur throughout the cell cycle. After accurate repair the
sequence can be re-cleaved and repaired again.
 It may not be necessary to have any non-homologous target sequences
to generate knockouts.

18. Working of NHEJ

19. Application of Genome Editing
o An effective technique that will allow scientists to adequately edit genes to
cure diseases. The case is similar for plant species.
o Where scientists desire to knock‐out a gene that will result to increase a
particular nutritional content.
o Sickle cell anemia is a great example of a disease in which mutation of a
single base mutation (T to A) could be edited by CRISPR and the disease
cured.
o It is used in Human gene therapy for curing disease.
o It can be used to modify the gene of Agriculture crops and animals
o It help in Ecological vector control (Mosquito sterilization etc)
o It help in Programmable RNA targeting.

20. Conclusion
Undoubtedly, this process caught most attention for their
potential in medical applications and numerous other
biotechnological applications like crop editing, gene drives
and synthetic biology.
Despite the enormous potential that lies within the
CRISPR-Cas9 technology, further investigation is required
to make the system an applicable and safe tool for
therapeutically useful approaches.

21. References
• Mussolino, C., & Cathomen, T. (2013). RNA guides genome engineering. Nature
biotechnology, 31(3), 208-209.
• Sun N. Engineering of transcription activator-like effector nucleases (talens) for targeted
genome editing. University of Illinois at Urbana-Champaign; 2013.
• Cradick, T.J., Keck, K., Bradshaw, S., Jamieson, A.C. and McCaffrey, A.P. (2010)
• Zincfinger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs.
Mol Ther, 18, Zou, J., Mali, P., Huang, X., Dowey, S.N. and Cheng, L. (2011)
• https://en.wikipedia.org/wiki/Meganuclease
• http://sites.tufts.edu/crispr/genome-editing/
• https://igtrcn.org/talencrispr-mutagenesis-in-aedes-aegypti/
• http://www.bio-rad.com