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CRISPR/Cas9

CRISPR/Cas9

CRISPR/Cas9

Intellectuals, researchers and scientists have long dreamt of the ultimate genetic manipulation technique – CRISPR-Cas9 allows them to do just that.

CRISPR-Cas9 system is naturally present in the archaea and bacteria to protect from invading viruses and plasmids. It works through complementary RNA guided cleavage of the foreign DNA, which subsequently is repaired through either imprecise non-homologous end joining or template-dependent homologous directed repair systems. Compared to other gene-editing tools such as ZNS (zinc finger nucleases) and TALENS (transcription activator-like effector nucleases), CRISPR-Cas9 is cost-effective, precise, reliable, robust, and multiplexed gene-editing tool. This Ebook gives a brief background into the basic mechanism of the CRISPR-Cas9 system and factors influencing its efficacy in treating human diseases, of its applications of agriculture, genetic engineering and environmental sciences. It also discusses various strategies and modifications to negate the off-target effects in CRISPR-Cas9 to improve the efficiency, safety and reliability as a weapon in the treatment of human diseases.

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MECHANISM OF
CRISPR-CAS SYSTEM

A CRISPR array is found within the bacterial genome. This array contains numerous unique protospacer sequences that have homologous sequences to certain foreign DNA. These protospacers have short palindromic repeat sequences between them. Below is an overview of the endogenous Type II bacterial CRISPR system:

01
In order to create the pre-CRISPR RNA (pre-crRNA), transcription of the CRISPR array is required
02
Via the activity of a special trans-activating crRNA (tracrRNA), the pre-crRNA is processed with homology to the short palindromic repeat. This tracrRNA contributes to the recruitment of RNAse III and Cas9 enzymes. Together, these separate the different crRNAs
03
The Cas9 nuclease and the tracrRNA make a complex with each crRNA.
04
Each of these complexes (crRNA-tracrRNA-Cas9 complexes) finds its complementary DNA sequence. The DNA is specifically complementary to the crRNA part of the complex.
05
Cas9 facilitates the separation of the double stranded DNA target after the complex binds and it cleaves the two strands close to the PAM.
06
After the double-stranded break, the crRNA-tracrRNA-Cas9 complex unbinds.

The CRISPR-Cas9 system includes the RNA-guided nuclease Cas9, which bonds to particular DNA sequences, complementing the RNA-guide sequence, and results in double-stranded breaks (DSB) on the DNA. The use of homology-directed repair (HDR) or non-homologous end-joining (NHEJ), means the double strand DNA breaks can be repaired. The application of this principle means that Cas9 and the guide-RNA have been altered in various ways that improve this system’s specificity and efficiency, growing its potential for several applications. This mechanism can be applied to point mutations, insertions, deletions, and sequence inversions to modify genetic loci.

Recent changes in the process make it a regulator of the genome, through the tethering effect or domains  to  the  Cas9  or  guide-RNA,  and  as  a  tool  for  visualization  by  binding  with  marker molecules. This multiplex scope of engineering CRISPR-Cas9 made it possible for scientists to relate this system for genome modifications in a wide range of organisms.

The CRISPR-Cas9 system is a powerful technology, applied in innovative and diverse biological applications.  Its  advantages  are  incomparable  to  other  gene  editing  tools.  The  CRISPR-Cas9 system has, for example, more target sites than TALENs and ZFNs, and Cas9 has several variants that can be applied in a wide range of studies. Furthermore, the ease of the use of the system means it can be carried out in just about any laboratory. Cas9-based tools have considerably improved our ability to conduct systematic analyses of gene function, and more accurately reproduce tumour-associated chromosomal  translocations.  This  technology  has also  created the  way forward  for epigenetics, the dissection of redundant gene functions, and potential gene therapy.  CRISPR-Cas9 technology is  currently the  most  versatile,  accurate and  simplest  genome editing  method in  a variety of in vitro and in vivo cells and organisms.