APPLICATION OF GENOME EDITTING IN LIVESTOCK
1.N.Kumari 2. N.Singh 3. P.Ghosh
1 First and corresponding author. Mail-I.D.-drnandanikumari@gmail.com Assistant Professor, Department Of
Animal Husbandry, R.V.C., B.A.U.
2. Batch topper, merit scholarship holder, 5th Year, R.V.C., B.A.U.
3. Merit Scholarship Holder, 3rd Year, R.V.C., B.A.U.
1. INTRODUCTION
Gene editing are the collective term used for a group of technologies that help the scientists in changing an organism’s DNA by means of addition. Removal, alteration of genome at particular location.
Genome editing is a further improvement over genetic engineering in which genetic material was randomly and non specifically inserted into a host genome which in case of genome editing, the editing and insertion sites area at specific locations. It is of vital importance for prevention and treatment of human diseases and in that direction, researchers are going on globally especially to understand diseases using cells and animal models.
According to a website, synthgo.com, pregene editing era consisted of genetic engineering which was revolutionized by two main events that took prior to the creation of recombinant DNA ( rDNA), First was the discovery of double helix DNA as discovered by James Watson and Francis Crick in 1953 Followed by Francis Crick in 1953 followed by the discovery by Arthur Kornberg in 1953 who was working on the project of DNA synthesis from about early 1980’s and he was finally able to synthesize DNA in vitro for the first time. He was awarded Nobel prize for his achievement. The Following information ( Source www.synthego.com) as found from the site may be enlisted as follows:-
1. 1970- Purification of Type II Restriction enzymes
2. 1971-Gene splicing Experiment paves the way for recombinant DNA (rDNA)
3. 1972- Creation of Recombinant DNA( rDNA)
4. 1974- Asilomer conference and temporary moratorium
5. 1975- Hybridoma Technique
6. 1981- Transgenic rabbit created using DNA microinjection
7. 1983- Discovery of PCR by Kary Mullis
8. 1985- Discovery of Zinc Finger nucleases ( ZFN)
9. 1990-2003: mapping of Human genome
10. 1993- CRISPR discovered
11. 1996- Cloning by Dolly the sheep
12. 1996- Sequencing of First human chromosome no. 21.
13. 2001- First gene targeted Drug therapy is approved with Glivec ( imanitib) an anticancer drug to treat myelogenous leukemia.
14. 2003- Glo Fish- Genetically altered fish
15. 2006- Pluripotent stem cell technology
16. 2010- World’s first synthetic life mycoplasma mycoides created
17. 2011- TALEN invented
18. 2012- Biochemical mechanism of CRISPR technology
19. 2013- Utility of CRISPR demonstrated in eukaryotic cell.
20. 2014- Concept of CRISPR gene given
21. 2015- CRISPER was used to edit human embryo
22. 2017- Two CAR T-cell therapies were approved
2. PRINCIPLES OF GENE EDITTING ( Jiang and Shan 2018)-
It includes Zinc Finger Nuclease, Transcription Activator like effector nuclease and Clustered Regularly Interspersed Short Palindromic Repeats ( CRISPR)/ CRISPR associated protein 9 (Cas 9).
1. ZINC FINGERS- They are the eukaryotic proteins motifs that facilitate specific interactions between proteins and nucleic acids or different proteins. The basic principle behind this technique is that each Zinc Finger specifically recognizes the consecutive nucleotides ( triplets) on a single DNA chain. It is a first generation technique for gene editing. In this a fused nuclease comprising a zinc finger protein ( ZFN) and a FOK 1 restriction endonuclease ( kim et.al. 1996).
In this technique, first the ZFP recognizes and binds to a specific DNA sequence. Then the dimerized FOK I, which possesses endonuclease activity cuts the recognized sequence. Due to this a DNA double stranded streak ( DSB) is formed which in due course is repaired through non- homologous recombination ( HR) ( Heyer et.al. 2010). In this process of repair of cell that ensues, mutation such as gene deletions or insertions are introduced which helps in accomplishing the task of gene editing.
2. TALEN TECHNIQUE-
After ZFN, Talen technique was discovered. It is a second generation editing technique. Just like the ZFN, its core component is a DNA binding protein and FOK I restriction endonuclease which unlike ZFN utilizes transcription activator like effector (TALEN), instead of ZFN to fuse with the FOK I RE. The TALE protein is a protein secreted and derived from Plant bacterial pathogen Xanthomonas ( Bonas et.al. 1989). It regulates endogenous gene expression in host cell due to structural similarity to transcription factor. It is basically made up of three components : an N-Terminal sequence for single transduction, a tandem repeat sequence and a C-terminal sequence that contains a nuclear localization signal and transcription activation domain. The important part of this TALEN is the tandem reoeat sequence contains 12-33 repeat elements having 12th and 13th amino acid as highly variable and referred to as repeat variable di-residue ( RVDs)
This RVDs region is very important as it determines the specificity of TALEN and are critical for targeting DNA. The four common RDVs namely NI has correspondence with A, NG has correspondence with T, HD has correspondence with C and NN has correspondence with G where A, T, C and G are respectively Adenine, Thymine, Cytosine and Guanine.
CRISPR-CAS 9
CRISPR Cas9 Gene editing:
CRISPRs are clustered regularly interspaced short palindromic sequences made up of an AT rich leader sequence followed by short repeats that are separated by unique spacers.It is an RNA mediated nuclease system adapted from the natural defence mechanism of bacteria and archae.The essence of crispr lies in the way it finds out a specific bit of DNA inside a cell. It was first time discovered in Streptococcus pyogenes.
How the defence mechanism of bacteria works?
Upon entry of a virus,the bacteria captures snippets of DNA from the invading virus and use them to create the spacers as a memory such that each time a new virus invades, the DNA segments are added on to the crispr array,this happens because protein complex Cas1-Cas2 identifies invading viral DNA and cuts out a specific DNA segment and this segment now known as protospacer gets inserted into front of crispr array , now if the viruses attack again there occurs transcription of long crispr RNA from repeats and spacers of crispr array ,cas 9 protein gets linked to it and cuts viral DNA and destroys the phage .Thus this is a unique idea that there are RNA segments having complimentary strands of DNA so once a DNA- RNA binds the target,the Cas9 nuclease enzyme degrades the DNA segment selectively and not the RNA.
But how does Cas9 distinguish between itself and viral DNA ?
This is possible because of PAM( Protospacer adjacent motif). It is a sequence of nucleotide having 2-6 bp which follows Protospacer sequence in viral genome. For eg. In Streptococcus pyogenes cas9 recognises PAM sequence GG ,whereas the spacer sequence in CRISPRs array are followed by GTT and not GG,so in this way the Cas9 isn’t able to bind to crispr array and avoids cutting its own genome and thereby is able to distinguish between the bacterial genome and viral genome.
3. APPLICATIONS OF GENE EDITING
MSTN( Myostatin) mutated Meishan Pigs using ZFN Techniques have 11.62% higher lean meat production
GFG5 ( mutated cashmere goats had significantly higher cashmere yield ( Wang et.al. 2016 a)
Burhard et.al. used CRISPR/ CAS 9 to delete a small sequence of the CO163 gene in pigs genome to generate 32 gene edited pigs.
Wu et.al. successfully utilized TALEN technology to insert a mouse SP110 gene into genome of Holstein Friesan cattle. Transgenic Cattle with SP110 gene knock in showed increased resistance to Mtb infection.
A BLG( Beta lactglobulin gene) leding to milk allergy in infants , was the gene to be removed and a knockout Cow was generated by Yu et.at. ( 2011) using ZFN technique to reduce BlG antigenicity and immunogenicity.
Lie et.al. 2018 tried to insert Fat 1 gene from coenorhabditis elegans into porcine Rosa 26 ( PRosa 26) locus in the CRISPR / CAS 9 system to successfully generate fat knock in pigs with increased n-3PUFAs.
With combined use of invitro fertilization and embryo transfer techniques, two hornless dairy cattle have been bred to avoid the painful horn cutting to improve animal welfare ( Carlson et.al. 2016)
To reduce the incidence of lethal or genetic diseases, it has immense potentials.
To increase disease resistance in crop and productivity, it is an important tool.
CONCERNS AND CONSTRAINS-
General public is yet not familiar and hence there is reduced acceptance of gene-editted organisms.
Ethical concern includes the misuse of this technology to produce organisms and products which could harm the mankind.
It might become a privilege of rich who would use these techniques to knockout their harmful genes
It is too early to predict that gene editing will not have detrimental effect on other genes.
4. CONCLUSIONS:-
To sum up it might be mentioned that gene editing is a boon to mankind for treatment, prevention and cure of some incurable, lethal or genetic diseases. It must be within the framework of set of some well defined rules to prevent it from becoming an ethical concern or a bioweapon or a luxury fad of the privileged.
5. REFERENCES-
1. www.synthego.com
2.https://en.m.wikipedia.org/wiki/CRISPR.
3.https://www.livescience.com/58790-crispr-explained.html.
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