Enhancing Homology Repair-Mediated Genome Editing

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park

Bill Reid, Ph.D. Postdoctoral Researcher, Postdoctoral Researcher, Mosquito and Fly Research Unit, USDA/ARS, Center for Agricultural, Medical and Veterinary Entomology, Gainesville, Florida MORE ABOUT THE AUTHOR

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.

Figure_1

Cas9 interacts with the strand complementary to the PAM (NGG) containing strand, leaving the PAM-distal target strand accessible to invading ssDNA.

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%).

http://igtrcn.org/wp-content/uploads/gravity_forms/9-c962e522ad861e8e9d396a8eb41308e3/2016/01/Figure_2.jpg

Branch migration occurs following the initial strand invasion of the 3′-end of a donor DNA molecule. This leads to the formation of a Holliday junction, which shifts along the donor/recipient DNA molecules analogous to a zipper. The growing recombination intermediate is called branch migration.

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.

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