How Crispr Cas9 is Used In Gene Editing

What is CRISPR?

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 ( Clustered Regularly Interspaced Short Palindromic Repeats).

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.

How Crispr Cas9 is Used In Gene Editing
How Crispr Cas9 is Used In Gene Editing
Originally the adaptive immune defence mechanism used in bacteria, for degradation of foreign genetic material, CRISPR has two components;

 1) A guide RNA and
 2) The Cas9 endonuclease.

Working mechanism:

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.

Types of cas9 genes used:

Till 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.

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, 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.

Specialities of Cas9 Null Mutant:-

 While the cas9 Null Mutant does not introduce mutations or directed recombination to the target genome, it offers great potential in genome targeting and can be used for the following:

1.Transcriptional Activation:  By Fusing the Cas9 Null Mutant with a Transcriptional Activator such as VP64.

2. 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

3. For DNA Labelling:  This is done by Fusion of the Cas9 Null Mutant with Florescent tags for genome imaging.

4.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.

Applications of CRISPR:-

  1.  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.
  2. The CRISPR /cas9 system offers the first alternative to the current protein-based genome editing techniques such as zinc finger and TALEN.
  3. The simple and effective mechanism of CRISPR is considered the game changer in molecular genetics and has been applied to many scientific fields.
  4. 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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