In a recent paper in Nature Biotechnology, Richardson et al (2016) report on the interaction of target-site bound Cas9 and dCas9 on DNA strand accessibility- and have identified an optimized approach to increase homologous recombination (HR) mediated repair.
They found that Cas9 remained bound to the DNA for ~5.5 hours following DNA binding/cleavage, and that the PAM-distal nontarget DNA strand was accessible to ssDNA invasion even when the Cas9 ribonucleoprotein complex was still bound to the DNA target. The success of the invading DNA was shown to be strand specific, where the invading ssDNA strand was identical to the Cas9 target strand. This pushed the PAM-distal nontarget strand out of the RNP complex through branch migration.
Using size optimized ssDNA donors as the invading strand that had 36 nucleotides matching the PAM-distal target site and 91 PAM-proximal nucleotides they edited a BFP reporter into a GFP reporter and achieved a whopping 57% success rate for HR-mediated repair in human cell culture. This demonstrated that this approach to HR-mediated repair is highly efficient for editing small regions of DNA.
Of additional interest was that this same HR-mediated genome editing approach could be applied using either the Cas9 nickase or the catalytically inactive dCas9, albeit at much lower frequencies (~1%).
Genome editing without the requirement of a double stranded DNA break has obvious implications for the treatment of disorders in human disease, but could also be applied for genome editing in insects that have extremely long life spans, for which off-target cleavage effects would be highly undesirable as they could not be easily out-crossed.
The authors further suggest that the donor strand bias may be a phenomenon specific to Cas9-mediated genome editing and that since their approach was independent of other tactics to enhance HR-mediated repair (eg/ knock-down or inhibitors of non-homologous end joining repair enzymes) that the efficiency of this approach to small locus HR-mediated repair could be increased when additional strategies are included.
Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. 2016. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. doi:10.1038/nbt.3481.