Clustered design process. One needs to only insert

Clustered regularly
interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have
proven to be one of the most versatile tools for genome editing.. CRISPRs are
genetic elements which immunize bacteria
against viruses. The protection mechanism includes 3 main stages: adaptation,
transcription, and interference. At the
adaptation stage, when foreign DNA entered bacterial cells, a small fragment is
incorporated into the CRISPR locus of the host genomic region in specialized
repeat structures separated from each other by short palindromic repeats and
therefore they received the name CRISPR. The Cas genes (CRISPR associated), are
localized very close to the CRISPR cassette express protein and have helicase
and nuclease activity (Haft et al., 2005).
At the transcription stage, the entire CRISPR locus is transcribed into a long
pre-crRNA (poly-spacer precursor crRNA) and produce short crRNAs (CRISPR RNA)
of 39–45 nucleotides containing one spacer sequence. The ability to provide
defense against invading genetic elements seems to render CRISPR/ Cas systems
particularly desirable in hostile environments (Godde and Bickerton 2006). Cas9 cleaves both strands of the target DNA
and binding specificity is defined solely by the chimeric crRNA:tracrRNA
molecule. CRISPR has been adapted to create RNA directed genome engineering
tools by introducing some modifications,
including codon CRISPR has been adapted to create RNA directed genome
engineering tools by introducing some modifications, including codon
optimization for the Cas9 nuclease for adequate transcription in higher
eukaryotic cells, fused to nuclear localization signals (NLS). In addition, a
single chimeric guide sgRNA (crRNA tracrRNA duplex) is constructed under the
control of promoter type III instead of expressing 2 non-coding RNAs (tracrRNA
and pre-crRNA)

Within
this RNA, a stretch of 20 bases is complementary to the respective target site.
 The CRISPR/Cas system is especially
attractive because of its very simple design process. One needs to only insert
the desired DNA oligonucleotide into a vector construct for target site
selection, as specificity is solely defined by base complementarity to the
guide RNA (gRNA). The Cas9 protein does not require any re-engineering and has worked well for all of the target sites. By
contrast, the CRISPR/Cas effector module, which is involved in the maturation
of the crRNAs as well as in target recognition and cleavage, shows a far
greater versatility (Mohanraju et al., 2016).