Abstract
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated protein Cas9 has revolutionized gene and genome editing. This system was discovered in prokaryotes as a defence mechanism to protect themselves from viral predators via three basic steps of spacer-acquisition, Crispr RNA (crRNA) processing and interference. CRISPR-Cas display a prokaryotic adaptive immune system that memorize previous infection by integrating short sequence of invading genome-termed spacers. CRISPR-Cas9 has been developed into a simple, inexpensive and versatile tool for genome editing. During genome editing three players play major roles, Cas9, Guide RNA and desired DNA piece. In Sickle Cell Disease which is caused by single DNA base mutation CRISPR-Cas9 system can guide RNA to identify the mutated DNA, cut out mutated sequence and insert the corrected sequence by Non-Homologous End Joining (NHEJ) or Homology Directed repair (HR). This technology has been studied for cure of several more diseases like Huntington disease, HIV, Duchene Muscle Dystrophy etc. The simplicity of CRISPR Cas9 has resulted in widespread use of this technology in many fields such as in agriculture, healthcare and in biofuels. CRISPR’s list of potential benefits is a long one but technology also brings with it a number of limitations like off target activity, mosaic generation etc. Overall, this is a powerful technology which if used judiciously and cautiously can contribute to advances in fields of biology, agriculture and medicine.
Introduction
In 21st century scientist found the easy and convenient way to edit the genome sequence. Modification of the DNA was made easy with the technique known as CRISPR. CRISPR allows us to target at specific sequence in the DNA and perform the alteration in all the genes present in the genome of the organism. CRISPR is the sequence information present in the bacterial genome which is regularly interspaced and repetitive in nature. CRISPR system acts as the defence system in the bacteria which prevents the bacteria from the attacks by different viruses specially bacteriophage.
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CRISPR is the Clustered Regularly Interspaced Short Palindromic sequence. It consists of a
- Short Palindromic Sequences: These sequences are normally 20 – 40 bp in length. This system works better with the Palindromic sequences. Palindromic sequences are those have same base pairs sequence both from 5’ end of the one strand and from 5’ strand of the other strand i.e. they are complementary to each other. They are also known as inverted strands. Palindromic sequences are taken so that after the transcription of the DNA into mRNA the so formed mRNA is identical to each other.
- Spacer Repeats: These are unique in itself and are located in between the palindromic sequences. These are the sequences that join the palindromic sequences. Spacer DNA is the set of the DNA sequences that join 2 palindromic sequences together in the DNA. Scientist in 2000 believed that the spacer DNA matches with the DNA sequences of the viral DNA that attacks the bacteria especially a Bacteriophage.
- Cas proteins: Cas genes are associated with the DNA strand which further gets translated to the CAS proteins or CRISPR associated proteins. These are normally enzymes that are Helicases and nucleases in nature. Nuclease CAS enzymes cut the bonds between the nucleotides of the viral DNA. However, Helicase enzyme acts on the hydrogen bonds present in between the 2 strands of the DNA helix. CAS 9 is the most important among all the types of the CAS enzymes. These enzymes have the ability to cause double strand breaks together on both the DNA strands. Therefore, these are known as molecular scissors.
How does CRISPR system works in Bacteria?
Bacteria gets affected by a virus namely a Bacteriophage. Bacteriophage invades the Bacterial cells and gets attached with the cell membrane of the bacteria. Due to this attachment of the Bacteriophage the genome of the virus is transferred to the host machinery of the bacteria which then gets multiplied in the bacteria and overtake the genome machinery of the bacteria leading to its death.
However, with the help of the CRISPR system the defence mechanism in the bacteria kills the virus in 3 step mechanism which involves:
- Spacer Acquisition
- CsRNA processing
- Interference
CRISPR system is of 3 types namely:
Applications of CRISPR
There are many applications of the CRISPR technology some of the ongoing ideas for the applications of the technique are:
- Pet Breeding: This technique is used to remove any sort of genetic disorder in the pets in order to make them healthy. For example: Breed of Dogs named Dalmatians which are prone to the bladder stones due to genetic mutations. Dog breeders in U.S plan to fix this by using the CRISPR technique.
- Allergy Free foods: It has normally be found that some food items have certain proteins that may cause certain allergies in people For example: some people are allergic from wheat due to the presence of Gluten in it. Some labs in Netherlands are working on the removal of the gene responsible for the Gluten production in order to make them suitable of the celiac.
- Production of Decaffeinated coffee: Tropic biosciences is the U.K based company using CRISPR cas is creating a variety of coffee which is naturally decaffeinated by turning off the genes present in the coffee which produces caffeine.
- Greener Fuels: Using the CRISPR cas the biofuels are produced by algae. Company named as synthetic genomics have created the strains of the algae that produces 2 times the fat which is used for the biofuel production. Scientist worked by removing the genes that limit fat production. This company have now collaborated with the oil company named Exxon MOBIL to produce 10,000 barrel of Algae biofuel per day by 2025.
- Pest Eradication: CRISPR helps to control the number of insects which are used for the transmission of the infection causing insects in the ecosystem. Last year gene drives were organised by Imperial college, U.K. to stop the spread of the Malaria. They introduced the genes among the mosquitoes and ensured that when both the parents carry it, the females stop laying the eggs to control the production.
However, the use of Gene Drive was considered ethical so it was banned.
Summary
CRISPR is the gene editing tool which is useful for introduction of the required genes in the genome of the organism. It also is the type of immune system in the bacteria which protects the bacteria from viral attack. It requires a set of palindromic sequences and spacer sequences and different Cas proteins. There are 3 types of CRISPR systems namely TYPE 1, TYPE II, TYPE III with 3 steps i.e. Spacer Acquisition, CrRNA processing and Interference. These are the different steps that helps to protect the bacteria from the different viral attacks.
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