CRISPR CAS 9 AS A GENE EDITING TOOL A NEW ERA IN MOLECULAR BIOLOGY
The clustered interspaced short palindrome repeats (CRISPR) and its associated protein (CAS 9) are adapted from the prokaryotic immune system. CRISPR is a family of DNA sequence in bacteria that contain short fragments of DNA from viruses that have attacked the bacterium while CAS 9 is a CRISPR associated protein that target and cleave the invading DNA in a sequence-specific manner.
CRISPR locus components: including ‘spacers’ that separate the repeats in the CRISPR locus, which are short stretches of non-repeated DNA and spacers are basically derived from the invading viruses. ‘Leader’ is a conserved sequence associated with CRISPR loci, located upstream of the CRISPR with respect to the direction of transcription. ‘Photo spacer’ is the region of invading virus which is similar to the nucleotide sequence of the spacer in order to recognize and to block the replication of the virus.
CRISPR working components: CRISPR CAS/9 has been modified to edit genome. By delivering CAS 9 nuclease with a synthetic guide RNA into a cell, the cell genome can be cut at the desired location, allowing existing genes to be removed and add the new one. Synthetic guide RNA consists of Cr RNA and tracer RNA. Cr RNA locates the correct section of host DNA along with a region that binds with (generally hairpin loop form). Tracer RNA bind to Cr RNA to make complex active.
Potential applications of CRISPR CAS/9: CRISPR CAS/9 base editing of genes in plants for example in legumes Nitrogen fixing rhizoid have established a symbiotic relationship with legume family. Many host genes in legumes involve in the SNF (symbiotic nitrogen fixation). CRISPR as a gene editing technology has been used in legume plants to improve the legume-rhizoid interaction and guide the engineering of SNF pathway into non-legume crops to reduce the use of nitrogen fertilizer.
In Nicotiana benthamiana some genes like BEYDV, TYLCV, BCTV, MEMV are responsible for viral infection scientist by using CRISPR gene editing technique knock out these genes and plant develop resistance against the virus (Ali et al., 2015).
In Populous trichocarpa, the DD43 region is present that involve in the production of Hygromycin phosphotransferase scientist knock out this region and plant develop resistance to Hygromycin. (Li et al., 2015)
Chinese research develops TB resistant cow: Scientist uses CRISPR CAS/9 system to insert a new TB resistance gene- NRAMP1 into the genome of bovine foetal fibroblast which is a cell derived from female dairy cows. Result reveals that NRAMP1 gene has successfully integrated into the genome of calves. When they exposed to Mycobacterium Bowie (the bacteria that cause bovine TB) the transgenic animal shows increased resistance to the bacteria measured by standard markers of infection in the blood sample.
Ethical Issues in Genome Editing Using Crisper/Cas9-System
The balance of risk & benefits
Potential of gene editing
The benefit of risk balance
Concepts in risks benefits assessment
Methods to balance risks & benefits
Global ecological disaster
Effect on biodiversity
Change in phenotype
Effect on biosphere
Regulations for Consumers
Consumer protection is required
Proper Labelling requirement
Proper field trials
Proper commercial release
Tampering with nature by mixing genes among species
Violation of natural organism intrinsic values
Stress for plant
The indirect effect of GMO, S On society:
Changes in population mating structure
Alteration of competitive hierarchies
Changes in species number
Changes in the physical and chemical environment
The ethical concern of crisper/Cas9 to the human germline
The principle of germline editing
Benefits of germline editing
Problems of germline editing in humans
Purpose of germline editing
- The balance of Risk and benefits:
An important ethical issue is that benefits must be greater than risk. Greater attention should pay toward risk since it causes damage to the human beings and the environment. Genome editing by using this method may increase the risk of damage and bring changes in the genetic material of an individual as a result of the gene may flow among the other closely related species. By horizontal gene transfer, the genetic material could allow the engineered genes to move into population other than the target host with undesirable consequences. In most extreme cases there are more chances of risk change in genetic makeup of an individual leads toward the genetic contamination of economically important crops and cause the extinction of native species or other species of local importance and leading to the loss of diversity.
A report from International journal of biological sciences has found that Monsanto’s genetically modified organism I-e corn cause organ damage in rats and it has adverse effects on human as well. A molecular biologist scientist Gilles-Eric Saraland at the University of Caen clearly explained the adverse effect on kidney, liver, Heart and adrenal gland.
- Ecological disequilibrium:
Every species has a role in the environment. Due to change in the role ecosystem disturb. One species dominates and other wipes out. Better performance of genetically modified organisms has the negative impact on other. Engineered changes in growth rate, tolerance of physical and chemical factors can change the performance of GMO, s with respect to naturally occurring organisms.
As the often cited example is the potential of increased weed among herbicide-tolerant crops. Increased weed could have the negative impact on surrounding agricultural crops and on wild-type vegetation in nearby crops. Both impacts could have economic consequences either directly through the loss of valuable cropland or indirectly through the loss of services in the ecosystem.
- Regulation for consumers:
Use of crisper/cas9 technique makes more difficult to identify the organism in the lab and then regulate in the market. Food and drug administration in the US approve the GMO, S but it is not known how they will handle such an organism in the market. Properly deregulate the organisms in the market without proper observance is the major problem.
For many years transgenic organisms have been patented when they have an industrial use; also human gene sequences have been patented for clinical use; what has made possible an enormous growth of biotechnology. So it is the responsibility of the Government that proper guidelines should be made for the proper regulation of the genetically modified organisms and the law should apply to everyone equally. It is necessary to commercialize only safe products.
- Ethical and social issues:
The use of GMO’s has the serious concern with religious belief, the proper distribution of seeds to low-income farmers and product labelling, may give birth to social and ethical issues which can have serious consequences in the unique socioeconomic set-up in Pakistan. It is observed that the recently concluded international treaties on patent and intellectual property rights such as WTO coupled with the present level of investment by different nations in R&D in biotechnology will aggravate the situation by further widening the existing gap in the biotechnology capabilities of the developed and developing countries.
Tampering with nature by mixing genes with the species is also an issue that changes the intrinsic properties of the organisms. As a result of this plants or other organisms has to face the stress conditions.
- Indirect effects of GMOs on society:
The releasing of GMO, S in the society may have the adverse effect that may include the change in the population mating structure, alteration of species genetic makeup and modification of the physical and chemical environment. Such type of alteration may cause the change in the structure and size of the population and species number. Indirect effects are difficult to detect and predict because these effects cannot easily observe with the naked eyes but they have serious effects on the ecosystem as a result of which ecosystem disturb and the different problem arises in the society. As a result of this effect, there are chances of removal of wild-type species from the ecosystem.
- The ethical concern to human Germ line:
It is also a major concern that by using such type of modified organisms’ human germ line may also change. In the case of genome editing the sequence of the gene in individual changes, these change might be transfer to the next progeny as well.
Change in the genetic makeup of upcoming progeny may bring changes in the DNA of other population and responsible for different diseases as well so the basic need of the hour is that alteration in the DNA should be done under control condition and suitable way after proper testing of the organism. Genetically engineered organisms are mostly banned in Europeans countries because of their belief that they have the adverse effect on wild living organisms
Conclusion: The CRISPR can utilize guide RNA to effectively recognize and target foreign DNA and RNA destruction. It is the flexible and accessible tool for multiple applications like genome editing and modulation of gene expression.
The use of crisper/cas9 may cause many ethical issues not only in human but also in other living organisms and environment as a result of which there are more chances of risk assessment and safety issues that may lead to unsuitable conditions and cause genetic impairment.