TARGETGENE

Genome editing- a brief intro​

Genome editing is a technique to artificially modify a genome in a living cell. ​

The Genome

The genome is the sum of an organism’s inherited genetic material that determines an individual’s biological characteristics. The substance composing our genome is DNA. Our genome is organized into chromosomes throughout which our genes are distributed. Each gene encodes a specific protein in our cells that may be involved in a trait such as eye color, height, or susceptibility to a disease. Therefore a mistake in the correct DNA sequence may encode a defective protein which leads to a genetic disorder like Cystic fibrosis (CF) or Duchenne muscular dystrophy (DMD). These mistakes are called mutations.

During our lives our DNA is often damaged in the form of breaks in its structure. This happens naturally and can also be caused by factors such as radiation, for example, from long exposure to UV from the sun. Normally, our cells recognize damaged DNA and repair it. However, The DNA repair mechanisms are prone to mistakes – thus forming mutations. These mutations might be involved in developing disease such as cancer.

 

However, induced DNA breaks can be also utilized for therapy. Disruption, repair, deletion or replacement of a gene can be done by forming a DNA break in a desired position of a specific gene. 

Genome editing using nucleases 

Nucleases are natural or engineered molecular DNA scissors. Genome editing utilizes these nucleases to create specific double-stranded breaks. These molecular scissors can be represented by the enzyme FokI. The FokI nuclease is a two-part enzyme- the first is a DNA binding part that recognizes and binds one specific DNA sequence and the second a is a nuclease subunit that cleaves the DNA. Since the mid-90’s Scientists have found a way to detach the FokI nuclease subunit and use it to link to different proteins that bind and recognize new DNA sequences. This type of alteration is the basis for the field of genome editing using the first Zinc Finger Nucleases (ZFN). These proteins have the ability to mutate, delete, replace or insert genes in variety of organisms. Nevertheless, ZFN technology was difficult to employ and suffered from low success rates. 

Breakthrough was achieved by the development of a different family of proteins called Transcription activator-like effector nuclease (TALENs). TALENs are based on a series of amino acids repeats that dictate the DNA recognition sequence. Each protein repeat recognizes a single nucleotide on the DNA (A,T,G or C) which enables high specificity and large DNA binding domains.

Both ZFNs and TALENs require two FokI nuclease subunits at the target site to work together like two halves of a scissors. This type of co-dependence is called a dimer. Thus, as each nuclease contains one subunit, cleavage will only happen when two ZFNs or TALENs bind at sequences flanking the target site together. This dimerization enables genome editing to be highly specific.

A major hurdle in using  ZFN and TALENs is the need to design and construct a new pair of nucleases for each genomic position. This made the design of nucleases for new targets labor-intensive, prone to failure, and many researchers did not implement it in their research

TargetGene’s novel approach was to build a single universal protein that can be programmed by short sequences of RNA. This invention, was the first RNA guided nuclease designed for genome editing, and was solidified in a patent in 2011. In the following year, the CRISPR system which protects bacteria from bacteriophages was harnessed for genome editing (link to 1). Jennifer Doudna and Feng Zhang’s pioneering research has led CRISPR systems to revolutionize the field of biology since it enabled researchers to easily use gene editing on any new targets.

Nevertheless CRISPR systems, which evolved in prokaryotic organisms with tiny genomes, lack the specificity needed for editing the much larger genome of human cells, causing unwanted off-target mutations.

TargetGene identified this flaw and has built the most precise and versatile genome editing system available, while maintaining the simplicity of use.

CRISPR off-target
CRISPR off-target
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