genome editing is the emerging branch which includes the specific and precise engineering of genomic regions

2. Genome engineering
• Genome editing=Genome engineering
• strategies and techniques developed for the targeted,
specific modification of the genetic
information or genome of living organisms.
• The major advantages
– which uses more recent knowledge and technology,
– it enables a specific area of the DNA to be modified
– increasing the precision of the correction or insertion
– preventing any cell toxicity and offering perfect reproducibility.
• Genome engineering and synthetic genomics :most
promising technologies in terms of applied biological
research and industrial innovation.

3. General principles
• Editing the genome using
– Homology directed Repair(HDR)
– Non Homologous End Joining (NHEJ)
– Early approaches :modifying genetic sequences
using only homologous recombination.

4. Homology directed repair (HDR)
• Mechanism in cells to repair double
strand DNA lesions.
• The most common form : homologous recombination.
• Only used by the cell when there is a homologue piece
of DNA present in the nucleus
• mostly in G2 and late S phase
• Important for suppressing cancer
• maintains genomic stability by repairing broken DNA
strands
• Assumed to be error free because of the use of a
template

5. Non-homologous end joining (NHEJ)
• Repairs double-strand breaks in DNA in the absence of
homologous templates
• G0/G1 and early S-phases
• "non-homologous" : the break ends directly ligated
• "non-homologous end joining“ : Moore and Haber(1996).
• utilizes short homologous DNA sequences called
microhomologies to guide repair
• Imprecise repair when the overhangs are not compatible
• Lead to translocations and telomere fusion :hallmarks
of tumor cells.

6. NHEJ & HDR

7. NHEJ & HDR

8. Microhomology-mediated end joining
(MMEJ)
• Also known as alternative non homologous
end-joining (Alt-NHEJ):More error-prone
pathway
• A homology of 5 - 25 complementary base
pairs (microhomology) on both strands -align
the strands
• Ligating the mismatched hanging strands of
DNA, removing overhanging nucleotides, and
filling in the missing base pairs.

9. Microhomology-mediated end joining
(MMEJ)
• overhanging bases (flaps) and mismatched
bases on the strands are removed and any
missing nucleotides are inserted.
• Chromosome abnormalities and other
complex rearrangements

10. The pioneers….
• 1980s: Mario R. Capecchi and Oliver Smithies - HR
as a “gene targeting” tool ( inactivation or
modification)
• Along with Martin J. Evans: modification of the
mouse genome
• 2007 :Nobel Prize in Medicine for their work
Martin J. Evans Oliver Smithies Mario R. Capecchi

11. Genetically modified Mice
DNA modification in
embryonic stem cell culture
Inject the modified stem cells
into mouse embryo
Genetically modified mice-
lab models to study human
diseases

12. Genome
Editing
site-directed
endonucleases
ZFNs Meganucleases TALENs CRISPRs
rAAV
mediated
Genome Editing

13. Methods in genome engineering
• Insertion
– to obtain a new function
– to compensate for a defective gene
• Inactivation, or “knock-out”,
– fundamental research to know function of a gene
– to remove a persistent viral sequence from infected
cells
– in agriculture to eliminate the irritant or allergenic
properties of a plant.

14. • Correction
– to remove and replace a defective gene sequence
with a functional sequence.
– drepanocytosis (sickle cell anemia).
– improve the properties of a species without the
addition of foreign DNA.
Methods in genome engineering

15. Transfection by causing dsDNA breaks
• Molecular Scissors –DSB lead to NHEJ and HR
• Restriction enzymes
–1-10 bp restiction sites
–Short and palindromic- occur in several sites
–A genome surgery approach : higher
degree of accuracy and security- more
precise tools.

16. • Genome engineering RE
– recognize and interact with DNA sequences that
are sufficiently long so as to occur only once, with
high probability, in any given genome.
– The DNA modification precisely at the site of the
target sequence
– recognition sites are larger than 12 base pairs
– Meganucleases, zinc finger nucleases, and TALEN
fusions
Transfection by causing dsDNA breaks

17. Meganuclease-based Engineering
• Meganucleases : Endonucleases family which are
characterized by their capacity to recognize and cut large DNA
sequences (from 12 to 40 base pairs)
• best known meganucleases proteins in the LAGLIDADG family,
• Natural meganucleases :slight variations in recognition sites
• Two methods for creating custom meganucleases:
– Mutagenesis involves generating collections of variants
using a meganuclease
– Combinatorial assembly is a method whereby protein
subunits from different enzymes can be associated or
fused.

19. Zinc finger nuclease-based
Engineering
• Zinc finger motifs occur in several transcription factors.
• The zinc ion :important role in the organization of their three-
dimensional structure- located at the protein-DNA interaction sites -
stabilizes the motif
• The C-terminal part of each finger is responsible for the specific
recognition of the DNA sequence.
• The recognized sequences are short, made up of around 3 base
pairs, but by combining 6 to 8 zinc fingers whose recognition sites
have been characterized,
• It is therefore possible to control the expression of a specific gene.
• fuse a protein constructed with the catalytic domain of an
endonuclease -induce a targeted DNA break,

21. Transcription activator-like effector
nucleases (TALEN)
• Artificial restriction enzymes generated by fusing a
specific DNA-binding domain to a non-specific DNA
cleaving domain.
• The DNA binding domains, which can be designed to
bind any desired DNA sequence, comes from TAL
effectors
• Tal effectors consists of repeated domains, each which
contains of highly considered sequence of 34 amino
acids, and recognize a single DNA nucleotide
• Advantages in targeted mutagenesis: (1) DNA binding
specificity is higher, (2) off-target effects are lower, and
(3) construction of DNA-binding domains is easier.

22. ZFN & TALEN

23. CRISPRs
• Clustered Regularly Interspaced Short
Palindromic Repeats
• genetic elements that bacteria use as a kind
of acquired immunity to protect against
viruses

36. Crisper-Cas 9

42. Experiments in plants
• Targeted indels produced in Nicotiana
benthamiana (Nekrasov et al, 2013)
• Targeted mutagenesis of rice phytoene genes in
cultured protoplast( Shan et al 2013)
• Targeted deletions and substitutions in rice (Xu et
al 2014)
• Targeted mutations in inositol oxidase (inox) and
phytoene desaturase (pds) genes in wheat
(Upadhyaya et al 2013)
• Virus resistance in Arabidopsis and Cucumber