CRISPR/Cas9-mediated null allele production in mice is highly efficient, however, the generation of conditional alleles has proven to be more difficult.
- HDR efficiency is highly correlated with Cas9 cutting efficiency, thus can be improved by carefully designing and choosing sgRNAs.
- Use long ssDNA, rather than dsDNA, for significantly reduced off-target integration.
- While generating conditional alleles via the integration of LoxP sequence, use one long ssDNA is more efficient than using two short ssDNAs.