5’— ~60-nt 5’ homology-attP sequence-NNNNGG ~60-nt 3′ homology — 3’’
- PAM
- NNN corresponds to the 3-nt adjacent to the PAM site that remain following Cas9 cleavage.
- attP sequence (50-nt): GTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTAG
Single-stranded oligonucleotides (ssODNs) of 200-nt or more can be ordered from Integrated DNA Technologies and other companies. Do not have the oligonucleotide PAGE-purified, as this can introduce trace chemical contaminants that reduce embryo viability.