Ant to the recognition of self and non-self DNA because are
Ant to the recognition of self and non-self DNA because are presents only in the foreign DNA sparing the CRISPR mechanism to delete itself. Indeed, protospacer sequences incorporated into the CRISPR locus are not cleaved presumably because they are not next to a PAM sequence. This prokaryotic system has been adapted to be used in vitro, merging the crRNA with a part of the tracrRNA in a hybrid called guide RNA (gRNA). Twenty nucleotides at the 5′ end of theCas9 variants Cas9 is a bi-lobed architecture protein with the gRNA nestled between the alpha-helical lobe and the nuclease lobe. These two lobes are connected through a single bridge helix. There are two main domains located in the multi-domain nuclease lobe: the RuvC, which shares an RNase H fold structure with other nucleases in the retroviral integrase superfamily [23], which cleaves the non-target DNA strand, and the HNH nuclease domain, that has a -metal fold that comprises the active site, which cleaves the target strand of DNA [24]. The gRNA base paired with target ssDNA is anchored by Cas9 as a T-shaped architecture. The nuclease also consists of a recognition lobe (REC) that matches the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 target sequence in the host DNA. Several Cas9 mutants including REC domain deletion and residues mutations in the bridge helix (BH) domain have been tested to improve its efficiency and to find other useful “side effects”. REC and BH mutants show lower or none CBR-5884 site activity compared with wild type, which indicate these two domains are crucial for the gRNA recognition and stabilization of the whole complex. Normally Cas9 performs a double strand break in the target DNA site, while introducing a D10A or H840A mutation into the RuvC- or HNH-like nuclease domains results in the generation of a single cut [25] (Fig. 2). These mutants also known as Nickase have also been shown to be useful for genome editing. Nickase cut either the complementary or non-complementary DNA target strands, respectively, in vitro.Biagioni et al. Journal of Experimental Clinical Cancer Research (2017) 36:Page 3 ofFig. 1 Type II CRISPR mechanism of action. Foreign DNA is cut and acquired by Cas1 and 2 between CRISPR repeat sequences (a) forming PAMs. Then a RNA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28667899 Polymerase transcribes part of the CRISPR repeat and part of the PAM generating a crRNA (b) that hybridize with a tracrRNA and reach a homologous target sequence on the genomic DNA (c). Cas9 performs a DSB that it is repaired with a NHEJ causing indel mutations and so probably a premature stop codon (d)Off target effect risks and possible solutions Even if very efficient, this system is not completely immune to errors, so understanding the possible weak sides could be helpful to prevent all potential off-target effects. Recently, a number of studies have examinedpotential off-target sites that differ from one to six positions from the on-target site in human cells [26, 27]. To prevent these effects, it has been suggested that higher GC content at the RNA:DNA interface might potentially help to stabilize binding the hybridization, indeed highFig. 2 Cas9 structure. The alpha-helical lobe and the nuclease lobe composed by, RuvC and HNH domain. The D10A and the H840A mutations in these last two domains cause the loss of ability to perform a DSB making only a single nick per strand. These particular Cas9 are commonly called NickasesBiagioni et al. Journal of Experimental Clinical Cancer Research (2017) 36:Page 4 ofrates of mutagenesis have been observed for off-target si.