This biological diversity and evolutionary pathways are recognized by comparing organisms with available model organisms. There is a need to develop more genetic tools for approaching new species along with the model organisms. This is not an easy task and it is a great challenge for comparative biology.
REVOLUTION IN GENOMES EDITING
Advanced technologies revolutionized the field of biology and experimental science. Genetic tools help the biologists to understand development, physiology and behavior of living organisms. With the help of these genetic tools, we can determine diversity and evolution of organisms. There are two techniques that bring revolution in the field of biology are; gene knockdown and DNA sequencing. By the help of these techniques, we are able to study biological processes in living organisms and functions of various specific genes in diverse species. Arabidopsis, mice, Drosophila and zebra fish are most popular approaches in genetics on which there is a lot of experimental work have done in past. But that study is not enough because biological diversity is changing over time. This biological diversity and evolutionary pathways are recognized by comparing organisms with available model organisms. There is a need to develop more genetic tools for approaching new species along with the model organisms. This is not an easy task and it is a great challenge for comparative biology.
To meet editing tools development challenges, gene knock-down approach and DNA sequencing approach were introduced in 1990’s. Gene knock- down approach was developed on the base of RNAi. RNAi is small interference RNA. In this technique small RNAs are used to identify and degrade the RNA that is targeted. RNAi is a natural process that is present in eukaryotes. Eukaryotes use RNAi for the protection of genome against viruses and transposons. Scientists use this technique to degrade the mRNA of their interest and modify the genome. This application is widely used in the world. By using RNAi, we can knock-out our desired genes but this application has some limitations. For example, RNAi has off-target effects, toxicity problems and efficiency in delivery.
With the help of sequence modification techniques, we can manipulate coding sequences and regulatory elements. By using double-strand breaks, the efficiency of sequence modification (gene targeting) has increased. These enzymes are known as mega nucleases. These are naturally sequence specific endonucleases that are used for making double-strand breaks. Mega nucleases are genetic tools for gene targeting.
Triple-helix forming oligonucleotides:
Triple-helix forming oligonucleotides are used as genetic tool but their uses are limited because this tool is successful with only some sequences.
Zinc-finger nucleases are artificial enzymes made by combining of several zinc-finger domains. These domains recognize DNA binding sites, bind on these sites and cause cleavage. There is another domain that is FOKI engineered with Zinc-finger nuclease and increase its specificity.
There are two major drawbacks of ZFNs that are:
ZFNs are not able to target all the sequences.
Sequences of domains are not precisely defined and affected by the nearly present domains.
TALEN is the abbreviation of TAL effector nucleases. TALENs are enzymes that are engineered by the several tandem repeats along with FOKI domain.
TALENs have many advantages over ZFNs that are:
TALENs can target any sequence.
It has high targeting specificity.
TALEN is most efficient genetic tool.
Limitations in TALEN:
There are many limitations in TALEN system that are:
It is a laborious technique.
There are chances of off-target effects.
Its activity is affecting by the methylation process.
CRISPR is the abbreviation of Clustered regularly interspaced short Palindromic repeats. These are endonucleases with the high simplicity and high efficiency that cause revolution in the field of gene editing. CRISPR/Cas is most recent and popular genetic tool for precise alterations in genome. It has greater efficiency than other genetic tools. This mechanism is naturally present in bacteria where it is used to target the invading viral DNA and provide defense mechanism. In prokaryotes, CRISPR there is RNA from viruses that are associated with the Cas proteins and form CRISPR/Cas complex system. The viral sequences are present in CRISPR loci that provide immunity to the bacteria against viruses. In 2012, Jinek and his colleagues discovered CRISPR/Cas system in bacteria, Streptococcus pyogenes, having single Cas 9 protein and 2 RNAs.
Jinek described that these two RNAs can be combined in single gRNA that increase the efficiency of CRISPR/Cas system. The sRNA is actually sequence of 20 nucleotides. Its 5’ end binds with target DNA and its 3’ end is in-variable sequence and form complex with Cas 9. There is a PAM (Protospacer adjacent motif) sequence present downstream of sgRNA is responsible for the interaction of Cas 9 and target DNA that determine recognition specificity. After getting DNA target, cleavage occurs within gRNA three nucleotides upstream of PAM sequence.
CRISPR has many advantages over ZFNs and TALENs that are:
CRISPR has increased accessibility of gene targeting.
Even methylation has not stopped the activity of CRISPR.
Limitations of CRISPR System:
However, there are many advantages of CRISPR system over other genomes editing tools but there are some limitations in this system:
The major limitation in CRISPR/Cas system is that there are chances of off-target in this system.
CRISPR has short range of gene targeting.
Applications of Genome editing tools:
There are numbers of benefits that are gained by the use of genome editing tools. Some of the applications are:
These genome editing tools have number of applications in medical field for example a revolution has come in drug discovery field.
With the help of genome editing tools, biologists are able to eradicate harmful bacteria and develop new effective antibiotics.
In the field of agriculture, by using these genome editing tools scientists have developed such crops that are insects and pests resistant, drought resistant, salt resistant, high yielding and more nutritious.
Treatment of Genetic Disorders:
Scientists have modified the human genome by using different genomes editing tools and treat various genetic disorders for example sickle cell anemia.