Unfortunately, DSB induction has also been observed in sequences differing by 1 nt or more from the protospacer sequence and is at least partially due to a tolerance for spacer:protospacer mismatches 3, 5, 6, 7, 8, 9. In this process, genetic information is transferred from the template to the genome, allowing pre-designed gene modification as subtle as the substitution of a single base pair. Alternatively, the DSB can be repaired by homology-directed repair (HDR) when a single- or double-stranded DNA template is provided. Subsequent error-prone DSB repair can leave a scar that in many cases cripples the gene product. If this sequence matches a genomic sequence, the so-called protospacer, that is followed by an “NGG” ‘protospacer adjacent motif’ (PAM), a DSB is induced 3 base pairs (bp) upstream of the PAM 3, 4. The location of a Cas9-induced DNA double-stranded break (DSB) is specified by a 20 nucleotide (nt) sequence (called: spacer) in the crRNA which together with the tracRNA produces the guideRNA (gRNA) that forms a ribonucleoprotein complex with Cas9. Paramount to these developments are the unparalleled ease-of-use and high efficiency of engineered RNA-guided nucleases, chief among them Streptococcus pyogenes Cas9 ( spCas9, Cas9) 1, 2.
Recent advances in nuclease-assisted gene modification technology are transforming fundamental and clinical science. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site.
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The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity.
Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes.
To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break.
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