For the first time, scientists are able to precisely edit any part of the human genome
the way they wanted, thanks to the ground breaking technology called CRISPR.
Since its discovery, CRISPR has revolutionized genetic engineering with its ‘molecular
scissors’ that can selectively disable or change genes in human cells, providing promising
gene therapy treatments to cancer and inherited genetic disorders.
Originally the adaptive immune defense mechanism used in bacteria, for degradation of foreign
genetic material, CRISPR has two components; 1) a guide RNA and 2) the Cas9 endonuclease.
When gRNA and Cas9 are expressed in a living cell, the gRNA/cas9 complex is recruited to
the target sequence, which is directly upstream of the PAM sequence, through complementary
base-pairing of the gRNA to the genomic DNA.
Once the complex localises to the target DNA, cas9 cuts the desired region with extreme
accuracy resulting in a double strand break.
the Double Strand Break created by Cas9 is then repaired by the cells own repair mechanism:
1) non-homologous end joining DNA repair pathway is used in the absence of a repair template.
With this pathway the ends of the DNA are simply ligated back together, which usually
leads to the introduction of small insertion or deletion mutations that disrupt the reading
frame of the desired gene.2) Alternatively the homology directed repair pathway could
be utilized in the presence of a repair template.
This template will have homology to the flanking regions of the double strand break.
this method of repair is highly accurate and could be used to introduce specific nucleotide
changes into the targeted gene The non-homologous end joining mechanism could be utilized to
introduce random mutations, mostly in the form of insertion or deletions, and could
be used to knockout the gene of interest.
On the other hand homology directed repair could be used for gene knock-out, gene tagging,
specific mutations, knock-in’s, or for promoter studies.To date, three variants of the cas9
endonuclease have been adopted in genome-editing protocols.
First: The wild type cas9, that was introduced earlier, which can site specifically cleave
double stranded DNA Second: A mutant form of cas9, known as the cas9 Nickase.
The Cas9 Nickase has one of its molecular scissors disabled, resulting in the cleavage
of only one DNA strand.
And third: The cas9 Null mutant where both of the nuclease domains are inactivated.
However, it still retains its ability to bind to DNA based on gRNA specificity.
The most important parameter in genome-editing is targeting efficiency.
Since mismatches at the 5` end of the gRNA are tolerated, the use of the wild type Cas9
usually leads to unintended off target effects.
To overcome this, one can use two gRNA, as shown here, that are adjacent on the opposite
strands of the target site with paired Cas9 nickases.
Since a single-strand break, or nick, is normally quickly repaired through the homology directed
repair pathway, using the intact complementary DNA strand as the repair template, off target
effects of the Cas9 Nickase is minimized.While the cas9 Null Mutant does not introduce indel
mutations or directed recombination to the target genome, it offers great potential in
genome targeting and can be used for the following: First) Transcriptional Activation: By Fusing
the Cas9 Null Mutant with a Transcriptional Activator such as VP64.
Second) Transcriptional Repression: This is done by Fusion of the Cas9 Null Mutant with
Transcriptional Repressors or Using a gRNA against the Promoter Region of the desired
gene Third) For DNA Labeling: This is done by Fusion of the Cas9 Null Mutant with Florescent
tags for genome imaging And Finally) For Chromatin Immunoprecipitation: This is done by Fusion
of the Cas9 Null Mutant with an antibody epitope tag to facilitate the pull down of specific
genomic loci CRISPR was first shown to work as a genome editing tool in human cell culture
by 2012.
It has since then been used in a wide range of organisms including baker's yeast, zebra
fish, Fruit flies, nematodes, plants, mice, and several other organisms.The CRISPR /cas9
system offers the first alternative to the current protein-based genome editing techniques
such as zinc finger and TALEN.
The simple and effective mechanism of CRISPR is considered the game changer in molecular
genetics and has been applied to many scientific fields.
CRISPR/Cas9 system shows extensive applicability in our modern health care system.
It has the potential to become the platform in genetic therapeutics and personalized medicine.
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