CRISPR-Cas9 is a genome editing tool which is faster, accurate and cheaper than other DNA editing techiques. CRISPR or Clustered regularly interspaced short palindromic repeats are stretches interspersed identical repeat sequences and unique invader targeting spacer sequences. Cas 9 is a protein that act as molecular scissors and cuts the DNA.
CRISPR- Cas system contains palindrome repeats with spacer gene in between.These spacer repeats are not the same, each spacer contains short sequences of bacteriophage genome from previous infection. Along with the CRISPR the bacteria also has the Cas genes that have helicase and nuclease activity.
The CRISPR technology came into light when the researchers were studying the defense mechanism of bacteria against viruses. CRISPR sequences are derived from the viruses that have previously attacked the bacteria.The bacteria have stored these short sequences so that these sequences along with the Cas proteins can identify the viral DNA when the next attack happens and destroy them with the help of CRISPR-Cas system.So we can say that the CRISPR acts as a guide for the Cas protein to attack the bacteriophage DNA.
HOW CRISPR-Cas SYSTEM WORKS
When a bacteriophage attacks the bacteria , the CRISPR -Cas system gets activated and the Cas genes are transcribed and translated to produce the Cas protein. Along with this the CRISPR DNA segment will transcribe to produce CRISPR RNA(cr RNA) which will fit into the Cas protein and they together will attach to the bacteriophage DNA as crRNA has sites complementary to the bacteriophage DNA. The Cas protein will then destroy the viral DNA.This mechanism is adopted in case the bacteria already has the viral sequence from an previous infection.
So what happens when bacteria encounters a completely new viral infection, where the bacteria doesn’t have a spacer sequence that matches? This time also the bacteria is ready , it creates a different class of Cas protein, a type 1 class Cas protein and takes the bacteriophage DNA and copies it into the CRISPR system and also destroys the viral DNA.So that next time the bacteria will be ready for another attack from the same bacteriophage.
Adopting the technique bacteria use in encountering a bacteriophage , the scientists developed a genome editing technology using CRISPR-Cas system.The research was done in Streptococcus pyogenes by Jennifer Doudna and Emmanuelle Charpentier. The advantage of using this bacteria was that it had only one Cas protein that is the Cas 9. The Cas9 protein has a bi lobed structure with a pair of nuclease (it has 2 sites to cut DNA). It also has sites for two long RNA- crRNA and tracer RNA which holds the cr RNA in place.
The scientists modified this system
1.They added their own sequence of gene (which is complementary to the target they want to cut).
2.They joined the CRISPR RNA sequence with the tracer RNA sequence.
They created a CRISPR RNA- tracer RNA chimera.This sequence is called the guide RNA(g-RNA).Therefore the CRISPR RNA system consists of a guide RNA and Cas 9 protein.
HOW DOES THE CRISPR-CAS9 SYSTEM WORKS
The scientists created a CRISPR Cas 9 system with the guide RNA of their choice which is complementary to the genome they would like to cut. Once done they introduce it into the cell, the guide RNA will bind to the complementary DNA and the Cas9 system will nick both the strands of the ds DNA.So the DNA now is broken apart and the gene is non functional. Further two types of approaches can be adopted
- The cell will automatically try to repair the DNA and this may lead to mutation, inactivating the gene.This can help the researchers in understanding the function of the gene(in its absence).
- The researchers have also tried to add a gene sequence into the nicked DNA.Thereby causing a new gene to be expressed.
Another important component of the CRIPR-Cas 9 system is the PAM or Protospacer adjacent motif.It is a 2-6 base pair DNA sequence present only in the viral genome following the target sequence in the bacteriophage. Only if the PAM sequence is present in the viral DNA does the Cas9 nuclease identify and cleave the target viral DNA.This sequence is absent in the bacterial CRISPR genes , which is why the Cas 9 nuclease does not attack it’s own bacterial CRISPR gene.
One can say CRISPR is unarguably a more effective, easy and cheaper tool for gene editing .It can easily used to study the role of particular genes and can also come in handy in treating genetic disorders having the advantage to be implemented in the embryonic stage cells.