Cleavable donor‐assisted <scp>CRISPR</scp>/Cas9 system significantly improves the efficiency of large <scp>DNA</scp> insertion in <i>Physcomitrium patens</i>

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. patens). The IVC donor, which contains two Cas9 nuclease recognition sites flanking the homology template, enables the in vivo release of the linear template for homology-directed repair (HDR) when co-delivered with the corresponding CRISPR/Cas9 plasmid into protoplasts. In our experimental framework, two distinct sgRNAs and four different DNA inserts were evaluated. Compared with standard circular donors, IVC donors significantly enhanced the efficiency of CRISPR/Cas9-mediated precise insertion of 5.8, 7.5, and 11.1 kb DNA fragments at the PpPDV2-4 sgRNA target site, improving integration rates from 29.6 to 67.8%, from 15.0 to 72.0%, and from 12.1 to 65.6%, respectively. At an alternative sgRNA2 target site within the Pp6c18_3160 locus, the IVC donor also demonstrated a higher knock-in efficiency for a 7.4 kb fragment compared with the standard circular donor. This IVC donor-assisted CRISPR/Cas9 approach for large fragment knock-in represents a powerful tool for basic research and synthetic biology efforts in moss species. Moreover, this strategy may be potentially applicable to crops that are amenable to protoplast transformation and regeneration, facilitating the improvement of key traits.