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) Inactivation of a gene (knockout) Modification of a gene nonspecific more specific

METHODS OF GENOME MODIFICATION Two Methods Non-directed modification of genome Directed modification of genome nonspecific 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 an enzyme necessary for transposition

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: 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

Recovery of Double-stranded Break

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).

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 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

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. 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.

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/ne0000/113162400/103_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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