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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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