CRISPR Cas9 – A Brief Introduction

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. Please leave your Questions and comments below
and we will answer them as soon as possible. For more information please visit our website.
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