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Genome editing is the new focus in biotechnological and biomedical research. So,

What is genome editing?

Genome editing is a technique used to precisely and efficiently modify DNA of a cell or organism. An enzyme cuts the DNA at a specific sequence, and when this is repaired by the cell a change or ‘edit’ is made to the sequence. Genome editing can be used to add, remove, or alter DNA in the genome. By editing the genome the characteristics of a cell or an organism can be changed.

There are several different types of engineered nuclease used in genome editing. They all contain a nuclease part to cut the DNA and a DNA-targeting part to recognize the DNA sequence they cut. CRISPR-Cas9 is the most common, cheap and efficient system used for genome editing. So,

What is CRISPR-Cas9?

CRISPR refer to Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated endonuclease (Protein 9) which is work as a “molecular scissors” to cut and edit DNA in a cell. 

The location at which the Cas9 nuclease cut the DNA to be edited is specified by guide RNA, which is comprised of a crRNA component and a tracrRNA component, either individually or combined together as a ‘single guide RNA’ (sgRNA). A sgRNA can direct the Cas9 nuclease to cut the DNA at the specific site in the genome. Once the Cas9 nuclease molecular scissors make a cut in the DNA, the cell’s own machinery and other elements will repair the cut DNA specifically.

The CRISPR-Cas9 system was originally discovered in bacteria that use this system to destroy invading viruses. Using CRISPR system the bacteria snip out parts of the virus DNA and keep a bit of it behind to help them recognize and defend against the virus next time it attacks. Scientists modified this system so that it could be used in other cells.

CRISPR-Cas9 system is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications in gene function studying, stem cell engineering, gene therapy, tissue and animal disease models and antibody KO validation. 

TOOL

 

HOW it works?

The target-specific RNA (crRNA) and auxiliary trans-activating crRNA simplify as tracrRNA compose as a sgRNA (single guide RNA), the sgRNA direct the Cas9 nuclease to a specific genomic locus via base pairing within crRNA sequence and the target sequence. Cleavage only occurs when there is a protospacer adjacent motif (PAM) around the targeted sequence of the invading DNA, ensuring highly accurate targeting (Figure 1).

crispr cas9 2

crispr cas9 1

                                                  

Cas9 nuclease has two functional endonucleases: RuvC and HNH. It occurs the second conformational change when the Cas9 binds to the target gene locus, the nuclease functional region locates the reverse strand of the target DNA. Cas9-mediated DNA cleavage results in a double strand break (DSB) of target DNA (about 3 to 4 nt upstream of the PAM sequence).

Following DNA cleavage, the break is repaired by cellular repair machinery through non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms (Figure 2).