Table 2. Recommendations for targeted gene replacement.

• Flanking homology arms of around 2 kb

• Homology arms extending outside of the dsDNA break sites

• No regions of extensive DNA homology within the gene replacement area

• A single dsDNA break positioned at one side of the gene replacement helps avoid excised / inverted alleles

• If possible, test multiple sgRNAs for activity and specificity

• Circular plasmids (versus linear) for transient transfection

• Do not cleave plasmid targeting vector

• If using two dsDNA breaks, check genotypes for excisions and inversions