CRISPR/Cas9

CRISPR/Cas9

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

The CRISPR system is naturally present in most archaea and bacteria to protect them from invading viruses and plasmids. The functioning of the CRISPR system involves RNA-guided cleavage of the foreign double-stranded DNA, which subsequently gets repaired through either imprecise non-homologous end joining (NHEJ) or template-dependent homologous directed repair (HDR) pathways. Compared to other gene editing techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 is a cost-effective, precise, reliable, robust, and multiplexed genome editing tool. This eBook gives an overview of the CRISPR/Cas9 system, the mechanism of genome editing involved, the factors affecting its efficacy in the treatment of human diseases, and the applications of the CRISPR/Cas9 system in agriculture, genetic engineering, and environmental sciences. The eBook also discusses various strategies and modifications to negate the off-target effects in CRISPR/Cas9 to improve its 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 typically found within most bacterial and archaeal genomes. This array contains numerous unique protospacer sequences that have homology to specific foreign DNA. These protospacers have short palindromic repeat sequences between them. Below is an overview of the CRISPR/Cas9 system, an endogenous type II bacterial CRISPR system.

01
Upon spacer acquisition from invading DNA, CRISPR array is transcribed into pre-CRISPR RNA (pre-crRNA).
02
The trans-activating crRNA (tracrRNA), being complementary to the pre-crRNA forms an RNA duplex with the pre-crRNA. Within the RNA duplex, pre-crRNA matures to form crRNA by recruiting RNAse III. The RNA duplex containing crRNA with tracrRNA is also known as the single guide RNA (sgRNA).
03
The sgRNA complexes with Cas9 endonuclease to form the ribonucleoprotein complex that assists in target interception.
04
The ribonucleoprotein complex conducts target search and recognition based on the complementary base pairing of crRNA with the protospacer within the target DNA adjacent to the protospacer adjacent motif (PAM).
05
Upon binding of the ribonucleoprotein complex with the target DNA, the Cas9 facilitates the unwinding of the DNA double-helical strand at the target site and cleaves the double-stranded DNA at 3 base-pairs upstream of its PAM region.
06
After the double-stranded DNA cleavage, the ribonucleoprotein complex unbinds.

Genome editing using the CRISPR/Cas9 system involves the cleavage of double-stranded target DNA using RNA-guided endonuclease Cas9. The cleaved DNA strands get repaired via HDR or NHEJ pathways. This implies that Cas9 and the sgRNAsequences can be altered in various ways to improve the specificity and efficiency of the CRISPR/Cas9 system and thus enhance its potential for several applications. CRISPR/Cas9 system can be used to obtain point mutations, insertions, deletions, and sequence inversions to modify genetic loci.

Recent developments in CRISPR technology enable its use for precise genome regulation and interrogation. Simultaneously it finds application as an imaging tool to track genomic loci in real time. Thus, the multiplex scope of engineering CRISPR/Cas9 has 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 that finds innovative and diverse biological applications. It has several advantages compared to other gene editing tools. The CRISPR/Cas9 system, for example, can target more sites on double-stranded DNA than TALENs and ZFNs, and Cas9 endonuclease has several variants that find a wide range of applications. Furthermore, the ease with which the CRISPR/Cas9 system can be used means that 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 therapies. CRISPR/Cas9 technology is currently the most versatile, accurate, and simplest genome editing method to conduct in vitro and in vivo genome editing in cells and organisms.