Mechanisms of precise genome editing using oligonucleotide donors

• DOI: 10.1101/gr.214775.116
• Publication Date: 2017-03-30T00:40:32Z
• Authors: Yinan Kan; Brian Ruis; Taylor Takasugi; Eric A. Hendrickson
• Keywords: genomic DNA

The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence homology donors, these facilitate high-efficiency precise (PGE) via homology-directed repair (HDR) pathways. However, identity hierarchy HDR (sub)pathways leading to formation PGE products remain elusive. Here, we established a green blue fluorescent protein conversion system systematically characterize oligodeoxynucleotide (ODN)-mediated using Cas9 its nickase variants human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors central heterologies, ODNs generated short tracts Gaussian-like distributions. Interestingly, single-nick-induced ODN produced biased either mostly uni- or bidirectional depending on relative strandedness nick. Moreover, were physically incorporated into only bidirectional, but not unidirectional, pathway. lesions, unidirectional pathway preferentially utilized even though knock-in mutation theoretically have been converted by both Collectively, our results suggest ODN-mediated utilizes synthesis-dependent strand annealing single-stranded incorporation Both pathways generate Although utilized, work unequivocally establishes existence This extends paradigms HDR-mediated gene guidelines for