1. METHODS OF GENOME ENGINEERING: A NEW ERA OF MOLECULAR BIOLOGYBy
Ijaz Gul
(12-arid-1232)
PhD (Biotechnology)
Department of Biochemistry ,
PMAS-Arid Agriculture University Rawalpindi

2. CONTENTS Introduction Genome Modification Methods
Non-directed genome modification
Directed genome modification
Nucleases Used for Genome Editing
Delivery of Programmable Nucleases
Conclusion
References

3. INTRODUCTION
Genome engineering refers to the strategies and techniques developed for the targeted, specific modification of the  genome of living organisms
A genome contains all information about the development and functioning of a living organism

4. Why Genome Engineering?
To develop animal models of human diseases to find appropriate treatment
To study gene expression
Introduce heterologous genes into the genome for scientific and biotechnology applications
To cure hereditary diseases

5. Modification of Genome
In general, modifications of a genome comprise
Insertion of a foreign gene (knockin)
Inactivation of a gene (knockout)
Modification of a gene
nonspecific
more specific

6. METHODS OF GENOME MODIFICATION
Two Methods
Non-directed modification of genome
Directed modification of genome
nonspecific
specific

7. Non-directed Modification of Genome
Mostly by
Direct injection of linear DNA into the cells
Viral vectors
Transposons

8. Non-directed Modification of Genome Cont..
1. Direct injection of linear DNA
Is the simplest(uses physical methods of DNA transfer)
But not very efficient method
Often results in the insertion of multiple copies of a transgene that forms concatemers

9. Non-directed Modification of Genome Cont..
2. By viral vectors
Viral vectors are very useful for delivery of foreign DNA into cultured cells(somatic cells)
Drawbacks
While dealing with animals, viruses are not able to get through the pellucid zone of oocyte(germ cell)
Viral vectors can also have undesirable effects when used for gene therapy

10. Non-directed Modification of Genome Cont..
3. By transposons
Transposons are mobile elements capable of moving through a genome

11. Non-directed Modification of Genome Cont..
Transposons move through the genome via the mechanism of cutting and insertion or copying and insertion

12. Non-directed Modification of Genome Cont..
Transposase
DNA transposon consists of a gene coding for a transposase
Transposase is expressed(oval in figure)
It binds to the inverted repeats of a transposon
Leading to cut and paste of the gene
an enzyme necessary for transposition

13. Non-directed Modification of Genome Cont..
Transposon mediated gene delivery
Any gene can be inserted into a genome using
1. A vector bearing a gene of interest
2.Flanked by two inverted repeats
3. A gene coding for transposase

14. Non-directed Modification of Genome Cont..

15. Undesirable Consequences of Transposons Mediated Genome Editing
Insertion occurs randomly and not in a pre chosen DNA locus
Insertional mutagenesis
Misregulated expression
Transgene silencing

16. Directed Modification of Genome
Directed modification of a genome fragment is possible using homologous recombination
Donor DNA for such manipulations should contain:
long flanking sequences
homologous to the locus of insertion

17. Homologous Recombination

18. Problems With Homologous Recombination
The main problem is undamaged target sequence is inert
The recombination level increases only after the target gene is damaged
DNA damaging agents stimulate homology recombination

19. Consequences of Double-stranded DNA Breaks
DNA breaks are regarded by a cell as potentially lethal damage
Double-stranded break (DSB) can be recovered via two pathways:
Homologous recombination (HR)
Nonhomologous end joining (NHEJ)
Both HR and NHEJ, when used with programmed nucleases
Allow introducing changes in a given site of a genome

20. Recovery of Double-stranded Break

21. NUCLEASES USED FOR GENOME EDITING
Double-stranded DNA breaks can now be induced in a given DNA fragment by
Programmable DNA-binding zinc-finger proteins (ZF)
TALE (transcription activator-like effector)
CRISPR (clustered regularly interspaced short palindromic repeat) system/CRISPR-associated protein 9 nuclease (Cas9).

22. Zinc Finger Nucleases Restriction enzymes composed of:
“programmable” DNA binding domains and
“constant” endonuclease domain
Type IIS restriction nucleases, for example FokI(Flavobacterium okeanokoites)
recognize short DNA sequences
introduce a double-stranded break

23. Zinc finger Nucleases Cont…
The site of the break can be changed by changing the specificity of the DNA-binding domain.

24. Drawbacks
Genome modification using zinc finger nucleases is a time-consuming process
Most such proteins are not working
A protein that binds its target efficiently in certain conditions will not bind with the same efficiency under other conditions

25. TALEN
Transcription activator-like effector nucleases (TALENs) are a new generation of “programmable” restriction enzymes
TALENs are engineered by fusion of:
DNA-binding domains derived from TALE proteins
and a nonspecific FokI endonuclease domain
TALENs are more specific and less cytotoxic
One advantage of TALENs compared to ZFNs
is their lower cytotoxicity.
TALEN genes are easier to manipulate
a TALE protein is almost as large as a ZF but recognizes only one base instead of three.
Some data shows that TALENs have higher specificity,which means that they induce fewer off-target breaks
and stimulate higher frequency of homologous recombination compared to other nucleases, including CRISPR-Cas9

26. CRISPR-Casa9 System
The CRISPR-Cas9 system is an adaptive immune system of bacteria and archaea
A cell “memorizes” a genome sequence of a phage that has infected it
A fragment of heterologous DNA about 20 nucleotides (called a spacer) is taken and inserted into the genome of the bacterium or archaean to elongate the CRISPR cassette
Foreign DNA is chopped up

27. CRISPR-Cas9 System

28. Advantage
Specificity of this system is defined by the small guide RNA and not by the protein as in case of ZFNs and TALENs
Compared to ZFNs and TALENs has the ability to digest methylated DNA
Simple method
It does not require generation of complicated genetically engineered constructs coding for modular DNA-binding proteins.
The biggest disadvantage of the CRISPR-Cas9 system is probably the rather high frequency of undesirable DNA breaks in sites partially complementary to the guide RNA.

29. DELIVERY OF PROGRAMMABLE NUCLEASES
One of the steps crucial for overall modification efficiency is the delivery of nucleases into cells
Nucleases can be introduced into a cell as a vector coding for a protein, as in vitro transcribed mRNA or as protein
Generally, DNA is delivered into cells in culture by electroporation or liposome  

30. DELIVERY OF PROGRAMMABLE NUCLEASES Cont…
As zona pellucid of oocyte is a barrier to DNA delivery
Usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells
Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection
However its ability to cross the zona pellucid and mediated gene transfer is unknown

31. CONCULSION
Directed genome editing technologies have become simpler and more available to researchers.
Double-stranded breaks generated by specially designed nucleases facilitate the process of genome editing.
Zinc finger nucleases – the first representatives of this technology – have been developed and improved for 20 years.
Nevertheless, some aspects of these technologies, including efficiency, decrease of off-target mutations, constructs generation, and delivery can be improved.

32. CONCULSION
All modifications are aimed to increase the overall system efficiency and safety for therapeutic approaches for genetic diseases.
More precise genome editing without off-target effects will allow manipulating a genome of a living organism without serious consequences.
Programmable nucleases with improved efficiency and specificity will open a new era in biological research, medicine, and biotechnology.

33. References
Michele Munk1, Luiz O. Ladeira2, Bruno C. Carvalho3, Luiz S and A. Camargo3. Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes. Scientific Reports |6:33588 | DOI: /srep33588 ISSN , Biochemistry (Moscow), 2016, Vol. 81, No. 7, pp © Pleiades Publishing, Ltd., 2016.

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