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METHODS OF GENOME ENGINEERING:
A NEW ERA OF MOLECULAR BIOLOGY
By
Ijaz Gul
(12-arid-1232)
PhD (Biotechnology)
Department of Biochemistry ,
PMAS-Arid Agriculture University Rawalpindi
CONTENTS
• Introduction
• Genome Modification Methods
 Non-directed genome modification
 Directed genome modification
• Nucleases Used for Genome Editing
• Delivery of Programmable Nucleases
• Conclusion
• References
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
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
Modification of Genome
In general, modifications of a genome
comprise
Insertion of a foreign gene (knockin)
nonspecific
Inactivation of a gene (knockout)
 Modification of a gene
more
specific
METHODS OF GENOME MODIFICATION
Two Methods
Non-directed modification of genome
nonspecific
Directed modification of genome
specific
Non-directed Modification of Genome
 Mostly by
Direct injection of linear DNA into the cells
Viral vectors
Transposons
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
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
Non-directed Modification of Genome
Cont..
3. By transposons
• Transposons are mobile elements capable of moving
through a genome
Non-directed Modification of Genome
Cont..
• Transposons move through the genome via the mechanism of
cutting and insertion or copying and insertion
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
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
Non-directed Modification of Genome
Cont..
Undesirable Consequences of Transposons
Mediated Genome Editing
 Insertion occurs randomly and not in a pre chosen
DNA locus
 Insertional mutagenesis
 Misregulated expression
 Transgene silencing
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
Homologous Recombination
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
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:
1. Homologous recombination (HR)
2. Nonhomologous end joining (NHEJ)
• Both HR and NHEJ, when used with programmed
nucleases
• Allow introducing changes in a given site of a
genome
Recovery of Double-stranded Break
NUCLEASES USED FOR GENOME
EDITING
 Double-stranded DNA breaks can now be induced in
a given DNA fragment by
1. Programmable DNA-binding zinc-finger proteins
(ZF)
2. TALE (transcription activator-like effector)
3. CRISPR (clustered regularly interspaced short
palindromic repeat) system/CRISPR-associated
protein 9 nuclease (Cas9).
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
Zinc finger Nucleases Cont…
• The site of the break can be changed by
changing the specificity of the DNA-binding
domain.
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
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
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
CRISPR-Cas9 System
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.
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
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
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.
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.
References
http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/n
e0000/113162400/103_1_2.jpg
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: 10.1038/srep33588
ISSN 0006-2979, Biochemistry (Moscow), 2016, Vol. 81, No. 7,
pp. 662-677. © Pleiades Publishing, Ltd., 2016.
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