1. GENOME EDITING University of Sulaimany College of Education
Chya Bahadeen –Diploma

2. What is a genome editig ?
Genome editing is a technique used to precisely and efficiently modify DNA within a cell.
It can be used to add (insertion) , remove (delete) or alter DNA in the genome .
By editing a genome the characteristics of the cell or organism can bechanged .

3. How does gene editing work?
Genome editing tools have two features:
Recognize specific DNA sequences (i.e. specific genes or non-coding elements)
Cut DNA (“nuclease”), then a scar is left behind

4. How does it work? molecular “scissors Specific genomic site
(e.g. gene)
Double DNA strand break (DSB)
Natural repair mechanisms

5. Nucleases create specific double strand breaks (DSB) at specific positions and harness the cell’s endogenous mechanisms to repair the induced break by natural processes
The consequences of double-strand genome breaks are potentially lethal to living cells and are rapidly repaired by cells using one of two principal pathways that are conserved in plants and animals

6. 1- pathway None Homologus End Joint (NHEJ)
the DNA ends produced by the break are re-joined by the cell’s repair machinery in a sequence-independent manner (i.e. regardless of the sequence at each end).
This is known as non-homologous end-joining (NHEJ). NHEJ does not necessarily restore the original sequence as it (and similar pathways) produces an insertion or deletion (an ‘indel’), usually of a small number of nucleotides, in a way that cannot be controlled at present.

7. Repair double strand break in DNA in absence of homologus template
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Repair double strand break in DNA in absence of homologus template
Imprecise repair when overchange are not compatible
Utilize short DNA seuences called micro homologus to guide repair

8. 2-pathway, homology-directed repair (HDR)
is DNA sequence dependent and uses an additional matching piece of DNA to provide template information that allows the double-strand break to be repaired correctly.
HDR can also be used to add or remove a prescribed DNA sequence at the site of the double-strand break in a manner that can be controlled.
The balance between the employment of NHEJ and HDR repair pathways by a cell in particular contexts is not well understood and is an active area of research.

9. Mechanism present in cell to repair double strand DNA lesion
Only used when there is homologue piece of DNA present in the nucleus
Assumed to be error free becouse of use of the template

11. Schematic of gene editing tools

12. 1-Meganucleases
Meganucleases were the first targeted nuclease described.
sequence-specific nucleases (high specificity)
Large recognition sites (12-40bp).
present in many microorganisms and plants
Most specific naturally occurring restriction enzymes
Advantages
Easy to deliver to cells
less toxicity

13. (limitations) not widely used for genome editing
Time consuming
Difficult to redesign to new target cells
This method is highly cost

15. 2-Zinc Finger Nuclease (ZNF)
Engineered DNA bind protein
Highly targeted double strand break (DSB) within the genome
Manipulation of the gene
stimulate cell’s natural repair process
DSB are important to site-specic mutagenesis

16. Component of Znic finger nuclease
DNA clevage domain
DNA bindind domain
- Comprised of a chain of two finger
modules
DNA clevage domain
-coprised of domain of fok 1
Component of Znic finger nuclease
DNA clevage domain
A bindind domain
DNDNA bindind doDNA bindind DNA bindind domain
domain
main
DNA bindind domain
c-terminal
c-terminal
N- terminal

17. FokI is a member an unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence.

20. Applications of ZFNs Repairing mutations
Insertion of gene or fragment of DNA at specific site
Applications in medical sector
Gene therapy
Treatment of HIV

21. Zinc finger limitation
Obtaining functional ZFNs require an extensive and time consuming process
Toxic to host cells
Difficult to ZFNS to target any desired DNA sequence
Recently TALENs have rapidly emerged as an alternative genome editing tool to ZNF

22. 3-Transcription activator-like effector nuclease TALEN
Are restriction enzymes that can be enginneered to cut specific DNA sequence
They are made by fusing of
DNA binding domain ( tale effector )
DNA cleavage domain (a nuclease which cuts DNA strands )
Contain tandom repeats of amino acid motif that can recognise single base though two di-variant residues
Advantages
DNA binding specifity is higher
Off target effector is lower
Construction of DNA binding domain easier
DNA binding domain- tale
-nuclease
FOK1

24. TALEN - limitation
Selection of mutation variants of the TALEN N-terminal domain that are capable of binding to A, G, or C.

25. 4-Clustered regularly interspaced short palindromic repeat/Cas- CRISPR Cas 9
(CRISPR) sequences are a defining component of a prokaryotic adaptive immune system.
Bacteria express genomically encoded RNAs to guide nuclease cleavage to matching sequences of invading phage and plasmid DNA.

26. CRISPR/Cas -protection mechanism -3stages:
1.adaptation- spacer acquisition
2.transcription-Biogenesis
3.interference

27. A-Spaceracquisition-adaptation
a distinct sequence of the invading MGE called a protospacer is incorporated into the CRISPR array yielding a new spacer.
This event enables the host organism to memorize the intruder’s genetic material and displays the adaptive nature of this immune system

28. B-Biogenesis To enable immunity,
the CRISPR array is transcribed into a long precursor crRNA (pre-crRNA) that is further processed into mature guide crRNAs containing the memorized sequences of invaders
Trans-activate (tra)crRNA is required for the processing of the pre-crRNA.
TransRNA

29. C.interference
the In the last stage of immunity, tracrRNA:crRNA(sgRNA) duplex guides the effector protein Cas9 to introduce a double-strand break in the target DNA

30. How CRISPR-Cas9 Technology Works
CRISPR-Cas9 is a gene editing technology that uses a combination of
an enzyme that cuts DNA (Cas9, a nuclease)
(2) a guiding piece of genetic material (guide RNA) to specify the location in the genome.
the guide RNA targets and binds to a specific DNA sequence
the attached Cas9 enzyme cleaves both strands of DNA at that site
This cut can be used to insert, remove, or edit the DNA sequence.
The cut is then repaired and the changes incorporated).
Scientists can create a guide RNA corresponding to almost any sequence within an organism’s genome.
Advanced Gene Editing: CRISPR-Cas9

31. TranscrRNA+ crRNA( combine) = single guide RNA

32. PAM :
Protospacer
Adjucent
Motif
Is a 2-6 base pair DNA sequence immediatly follwing DNA sequence targeted
By cas9 nuclease in
CRICPR cas system.In case of case9 it is 5’ NGG 3’

33. it is much cheaper, compared to other tichnique more efficient
Why CRISPR over other gene editing tools?
CRISPR-Cas9 is considered to be superior to other gene editing tools.
it is much cheaper, compared to other tichnique
more efficient
Ease fo use .
A prospect of making edits at multiple sites in the genome in a single procedure.

35. Comparison between ZFN , TALEN , and CRISPR /cas
Properties
ZFN
TALEN
CRISPR/cas
Off target effect
Low
high
Time consumption
Long (7-15 days)
Long (5-7 days)
Short (1-3 days)
essential components
Zinc finger protiens + Fok 1 (fusion protein)
Tale + Fok 1
(fusion protein)
Giude RNA + case9
Protein
Efficiency
Poor 33%
High 70%
Cost
High

37. Refrences
1-Megan D. Hoban and Daniel E. Bauer, Y 2016 x VOLUME 127, NUMBER 21 , Review Series , A genome editing primer for the hematologist
2-Ori Chalom. Kelly McBride Folkers
,NYU LANGONE HEALTH,
GENETIC EDITING: ETHICAL AND SOCIAL ISSUES
3-Professor Jonathan Montgomery ,et al,
September 2016,
Nuffield Council on Bioethics,
Genome editing
4-Stephen L Hart,
Patrick T. Harrison,
December 2017
,A beginner's guide to gene editing
5-Emmanuelle Charpentie
Frank Hille
,August 2016,
.royalsocietypublishing,
CRISPR-Cas: biology, mechanisms and relevance