Mechanism of CRISPR-CAS System

Mechanism of CRISPR-CAS System

Like every living being bacteria and other micro-organisms also face continuous challenges in form of varying environmental factors and pathogens. The biggest enemy of bacteria is the bacteria eating viruses called the bactreriophages. And like every organism bacterial cell has also developed a way to deal with it. With the passage of time bacteria have developed a natural system, a kind of immunity to deal with the invading viruses, and this special system of bacteria is known as “CRISPR”.

CRISPR is a natural immune system embedded inside the genome of various micro-organisms till now about 40% of bacteria and almost 90% of the total archaea, whose genome has been sequenced, has been reported to have the CRISPR-CAS immunity which enables them to fight back the invading viruses and prevent the viral infection. In the beginning very little was known about this immunity system but with continuous advancement in the field of genomics and molecular biology much has been discovered about this system, what it is composed of and how this system works.

Components of CRISPR-CAS System:

The CRISPR-CAS system exists as a part of the bacterial genome which upon the entrance or attack of a virus gets trigged and start producing corresponding products which enables the bacteria to stop viral infection. The CRISPR region of the bacteria is composed of three parts:

1.  CAS genes
2.  Spacer DNA
3.  Palindrome repeats

Image showing 3 parts of CRISPR Locus

Each individual part of the CRISPR region plays its own unique role in order to achieve the overall activity of the CRISPR system.

CAS Genes— These genes encode for special protein called the CAS protein which plays a dual role in the CRISPR system it acts like a helicase as well as endonucleases and is responsible for the unwinding, cutting and ultimately denaturing the invading viral genome.

Spacer Region—The spacer region is important for the invading genome detection as it encodes for a specific type of RNA called CRISPR RNA (CrRNA), this CrRNA is loaded inside a CAS protein and used as a reference, wherever the CrRNA matches the invading viral DNA the CAS protein cleave it from that place and ultimately denatures the viral DNA . The Spacer region is in fact a bacterial archive in which it stores a large number of copies of previously attacking viral DNA’s, whenever a new type of virus invade the bacterial cell the bacteria produces a special kind of CAS protein called the CAS1-CAS2 protein complex which denature the invading viral DNA and stores a copy of it in CRISPR region. This makes future detection of the invading viral DNA’s even easier. 

Palindrome Repeats—The palindrome repeats separates individual spacer DNA’s from each other in the CRISPR region. They also encodes for a special kind of RNA called the tracrRNA. This tracrRNA acts as a backbone for the CrRNA and keeps it firmly placed inside the CAS protein.

Later in 2009 Jennifer Doudna and Emmanuel Charpentier combined the CrRNA and tracrRNA in to a single GuideRNA (gRNA). They also successfully replaced the CrRNA region with a desired sequence. These discoveries lead to the foundation of use of CRISPR-CAS System for genome editing in the world of biology.

Mechanism of CRISPR-CAS System:

The CRISPR –CAS system involves 3 stages:

1.  Adaptation/Acquisition  
2.  Expression
3.  Interference  

1.Adaptation/Acquisition:

Adaptation is the starting and most important stage of the CRISPR mechanism as it keeps the CRISPR-CAS system up to date and provides a wide range of defence against a large number of viruses. Whenever a virus attacks it injects its DNA in to the bacterial cell in response to it CRISPR locus becomes active and produces a complex of two special types of CAS proteins the CAS1-CAS2 complex. This protein complex interacts with the invading viral DNA, cleaves it, produce a copy of its segment and insert it inside the CRISPR locus as spacer DNA for future use. The new coming spacer DNA is always added at the beginning of CRISPR right next to the leader sequence thus creating a sequential record of viral infection. Another protein called the integration host factor(IHF) ensures accurate insertion of the new spacer in to the CRISPR locus.

Acquisition Process 

New Spacer added in to CRISPR locus

2.Expression:

During the expression stage, in response to invasion of viral DNA, the CRISPR locus activates and transcribes the CRISPR array to from a precursor CrRNA which is then further processed in to CrRNA which is then loaded in to the endonucleases for the interference stage.

3.Interfernce:

During the interference stage in the CRISPR-CAS System, especially the CAS-9 system, the mature CrRNA is loaded inside the endonucleases molecule supported by the tracrRNA. This protein-RNA complex interacts with the viral DNA, slides over it in search of special sequences called Protospacer Adjacent Motif(PAM) which lies right next to the DNA sequence complementary to the CrRNA. This results in double confirmation of the invading viral genome and as a result the endonuclease cleaves and denatures the viral DNA preventing the viral infection.

Conclusion:

The CRISPR-CAS System has a tremendous potential of interacting with different genomic molecules and producing desired alteration in it. This revolutionary genome editing technology has opened new horizons in the field of life sciences. Scientists are now working on:

•  Gene Therapy—treating diseases by gene editing in affected cells. E.g. USA performing trials for treating HIV by knocking out genes encoding for receptor sites on T-cells.
•  Gene Drive—some genes passes faster than others in the wild, efforts are being made to cure malaria by treating certain genes with genome editing tools to make it unfit for carrying pathogen.
•  Food and livestock modification—efforts are being made to create high quality and more productive plants and animals using genome editing technology as CRISPR edited plants are escaping the GMO regulations.
•  Human Germ Line—efforts are being made to alter the genome of human sperm and egg in order to prevent spread of genetic disorders.
• Designer organisms—this technology also opens a new way for the creation of organisms which are especially designed at each growth stage and are perfect in every manner. Organisms including humans.