Mechanisms of precise genome editing using oligonucleotide donors

Area of Science:

• Molecular Biology
• Genetics
• Genome Engineering

Background:

• Programmable nucleases like CRISPR/Cas9 have revolutionized genome editing.
• Precise genome editing (PGE) relies on homology-directed repair (HDR) pathways, but the specific mechanisms remain unclear.
• Oligodeoxynucleotides (ODNs) are increasingly used as donors for targeted genome modification.

Purpose of the Study:

• To systematically characterize oligodeoxynucleotide (ODN)-mediated precise genome editing (PGE) using Cas9 and its variants in human cells.
• To elucidate the identity and hierarchy of homology-directed repair (HDR) sub-pathways involved in ODN-mediated PGE.
• To establish guidelines for optimizing PGE in human cells.

Main Methods:

• Development of a green to blue fluorescent protein conversion system for monitoring PGE.
• Systematic characterization of ODN-mediated PGE using Cas9 and nickase variants.
• Analysis of conversion tract lengths and distributions, and ODN incorporation into the genome.

Main Results:

• ODNs generated short conversion tracts with Gaussian-like distributions, distinct from double-stranded DNA donors.
• Single-nick-induced PGE showed pathway bias (uni- or bidirectional) depending on ODN strandedness and nick location.
• ODNs were incorporated into the genome only via the bidirectional conversion pathway, with preferential utilization of the unidirectional pathway in the presence of double-stranded lesions.

Conclusions:

• ODN-mediated PGE in human cells involves synthesis-dependent strand annealing and a newly identified single-stranded DNA incorporation pathway.
• Both pathways produce short, Gaussian-distributed conversion tracts.
• This research clarifies HDR mechanisms and provides essential guidelines for precise genome editing in human cells.