CRISPR-Cas9 SYSTEM

CRISPR-Cas9 SYSTEM

It is a genome editing tool. It is currently one of the fastest and most accurate

technique of editing DNA. The CRISPR-Cas9 system is a simplified version of the

antiviral defense system seen in bacteria. It is of great importance in

biotechnology and medicine due to high precision genome editing available for

cheap.

The CRISPR-Cas9 system consists of two different molecules

1. CRISPR:

CRISPR is “clustered regularly interspaced short palindromic repeats”. It is a

family of DNA sequences found in the genome of prokaryotes. This is a

primitive form of secondary immunity in the bacteria as CRISPR retains

information of previous infections by bacteriophage viruses and detect and

destroy bacteriophage DNA during subsequent infections.

2. Cas9:

It stands for “CRISPR associated protein 9”. It plays an important role in

immunity of bacteria against plasmids and DNA viruses. It cuts the foreign DNA

that enters the bacterial cell and nullifies its pathogenicity. This endonuclease

action is used to cut the bacterial cell genome at specific points.

Mechanism

The CRISPR-Cas9 system has high fidelity and is simple to make. It is also highly

specific. This specificity is due to the presence of a target sequence and

Protospacer Adjacent Motif (PAM) on the host genome. These two

components allow for binding of Cas9 protein for its action.

For recognition of the target sequence and binding to host DNA, there is a

need of guide RNA. The guide RNA is called CRISPR RNA (crRNA). This crRNA

binds with tracrRNA (trans-acting CRISPR RNA). The tracrRNA, being partially

complementary to crRNA, forms a RNA duplex, which is the active complex

that is involved in guiding the Cas9 protein to the site in the DNA where editing

is necessary. In some cases, both crRNA and tracrRNA are packaged together

to give a single guide RNA (sgRNA).

The Cas9 protein recognizes the PAM sequence (to differentiate bacterial self-

DNA from foreign DNA, binds to the host DNA with the help of crRNA and

forms either a single or double strand break based on the variant of Cas9.

Delivery

The delivery of Cas9, guide RNA and other essential components into the host

cell is done by viral or non-viral vectors. This is called Transfection. Depending

on the type of cell, the difficulty of transfection varies. For most cells, chemical

transfection via lipids and peptides is enough to complete delivery. But for

some cells like stem cells and neurons, more efficient delivery systems are

required (eg: lentivirus, adenovirus and adeno-associated virus).

Applications

CRISPR-Cas9 system for treatment

1. CRISPR-Cas9 system has been proposed as a treatment for multiple

diseases like cancer, Hutchinson-Gilford Syndrome, Huntington’s

disease, Cystic Fibrosis, Duchenne Muscular Dystrophy etc.

Antibiotic resistance

2.CRISPR-Cas-based "RNA-guided nucleases" can be used to target genes

encoding antibiotic resistance. This provides protection of

bacteria against invading foreign DNA, like transposons,

bacteriophages, and plasmids. This was a strong

selective pressure for acquiring antibiotic resistance and

virulence factor in bacterial pathogens. It is seen to have limited

replication of different herpes viruses and also proven to be effective in

eradicating viral DNA of Epstein-Barr virus.

Ovarian cancer

3. CRISPR has shown effectiveness in treating glioblastoma and metastatic

ovarian cancer in animal models. As of now, only one clinical trial was

done in the US, which involved treatment of Lung cancer by

immunotherapy. It aimed to do so by extracting T-cells from the patient

and making 4 genetic modifications to the cells to better detect the

cancer cells and kill them more efficiently.