Cleavable donor-assisted CRISPR/Cas9 system significantly improves the efficiency of large DNA insertion in Physcomitrium patens.

Precise insertion of desired fragments can be achieved by CRISPR/Cas9-based genome editing. However, a decrease in knock-in efficiency has been observed with increasing length of exogenous inserts. In this study, we developed an in vivo cleavable (IVC) donor-assisted CRISPR/Cas9 system to improve efficiency, particularly for larger inserts, in the moss Physcomitrium patens (P.