The experimental approaches based on this multipurpose technology

The prokaryotic type II clustered regularly interspaced shortpalindromic repeats (CRISPR)-Cas9 system is rapidly revolutionizing the geneticengineering field, altering the genomes of large varieties of organisms withrelative ease. Cancer genetics can be changed using experimental approaches basedon this multipurpose technology 1.

Principal component of the Type II CRISPR/Cas system is theCas9 endonuclease, a prokaryotic adaptive restriction system against invadingnucleic acids, such as those of bacteriophages and plasmids. Recently, thisRNA-directed DNA endonuclease has been coupled to target DNA sequences ofinterest. Through guidance of a 20 nucleotide RNA (gRNA),CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairsupstream of a PAM (Protospacer Adjacent Motif) 2. Cas9 not only edits the genomes of a number ofdifferent prokaryotic and eukaryotic species, but also an efficient system forsite-specific transcriptional repression or activation 3.

It can modify any genomic sequences, therebyproviding a specific, simple, easy, and cost effective means of genome widegene editing, analogous to the search function in modern word processors, Cas9can be guided to specific locations within complex genomes by a short RNAsearch string. Cas9-mediated genetic perturbation is simple and accessible, exposingthe functional organization of the genome at the systems level and establishingcausal linkages between genetic variations and biological phenotypes 4 As a RNA-guidedDNA recognition platform, it permits precise, scalable and robust RNA-guidedtranscription regulation 5, in contrast to RNA-mediatedinterference (RNAi), that uses small interfering RNAs (siRNAs) or short hairpinRNAs (shRNAs), that act as sequence-specific gene suppression in eukaryoticorganisms 6 but also non-specificand inefficient 7. Genome engineering via RNA-guided CRISPR-Cas9 systemprovides a novel methodology, to induce genomic modifications under theendogenous gene promoters 8. During the last few years, the clustered regularlyinterspaced short palindromic repeats (CRISPR) and the associated Cas9nucleases (CRISPR-Cas9) have modernized the choices for targeted genome editing9.

These programmable RNA-guidedendonucleases (RGENs) comprise two RNA elements, CRISPR RNA (cRNA) and itstransactivating RNA (tracRNA), which fused together and induce a targeteddouble-strand break (DSB),providing a corresponding DNA template, any specificgene sequence can be introduced via homologous recombination (HR) 10. 

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