The Convenience of Single Homology Arm Donor DNA and CRISPR/Cas9-Nickase for Targeted Insertion of Long DNA Fragment.

Objective: 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 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs).

Results: 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 knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus.

Conclusion: 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.