Clustered design process. One needs to only insert

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Last updated: April 14, 2019

Clustered regularlyinterspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems haveproven to be one of the most versatile tools for genome editing.

. CRISPRs aregenetic elements which immunize bacteriaagainst viruses. The protection mechanism includes 3 main stages: adaptation,transcription, and interference. At theadaptation stage, when foreign DNA entered bacterial cells, a small fragment isincorporated into the CRISPR locus of the host genomic region in specializedrepeat structures separated from each other by short palindromic repeats andtherefore they received the name CRISPR. The Cas genes (CRISPR associated), arelocalized very close to the CRISPR cassette express protein and have helicaseand nuclease activity (Haft et al., 2005).At the transcription stage, the entire CRISPR locus is transcribed into a longpre-crRNA (poly-spacer precursor crRNA) and produce short crRNAs (CRISPR RNA)of 39–45 nucleotides containing one spacer sequence. The ability to providedefense against invading genetic elements seems to render CRISPR/ Cas systemsparticularly desirable in hostile environments (Godde and Bickerton 2006).

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Cas9 cleaves both strands of the target DNAand binding specificity is defined solely by the chimeric crRNA:tracrRNAmolecule. CRISPR has been adapted to create RNA directed genome engineeringtools by introducing some modifications,including codon CRISPR has been adapted to create RNA directed genomeengineering tools by introducing some modifications, including codonoptimization for the Cas9 nuclease for adequate transcription in highereukaryotic cells, fused to nuclear localization signals (NLS). In addition, asingle chimeric guide sgRNA (crRNA tracrRNA duplex) is constructed under thecontrol of promoter type III instead of expressing 2 non-coding RNAs (tracrRNAand pre-crRNA)Withinthis RNA, a stretch of 20 bases is complementary to the respective target site. The CRISPR/Cas system is especiallyattractive because of its very simple design process. One needs to only insertthe desired DNA oligonucleotide into a vector construct for target siteselection, as specificity is solely defined by base complementarity to theguide RNA (gRNA). The Cas9 protein does not require any re-engineering and has worked well for all of the target sites.

Bycontrast, the CRISPR/Cas effector module, which is involved in the maturationof the crRNAs as well as in target recognition and cleavage, shows a fargreater versatility (Mohanraju et al., 2016).

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