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 of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a

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... green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussianlike distributions. Interestingly, single-nick-induced PGE using ODN donors produced conversion tracts biased either mostly uni-or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells.