The Convenience of Single Homology Arm Donor DNA
and CRISPR/Cas9-Nickase for Targeted Insertion
of Long DNA Fragment
CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid.
Materials and Methods
In this experimental study, single homology arm donor was applied
along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its
mutant nickase variant (Cas9n). Using
Both wild type Cas9 and Cas9n could conduct the knock-in process with this system.
We successfully applied this strategy with Cas9n for generation of
While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.