The Plant journal : for cell and molecular biologyFeb 21, 2025

A removable donor method improves large DNA insertion efficiency in Physcomitrium patens using CRISPR/Cas9

Updated Mar 3, 2026

Abstract

IVC donor-assisted CRISPR/Cas9 improved integration rates for DNA fragments up to 11.1 kb in the moss Physcomitrium patens.

A decrease in knock-in efficiency is associated with the increasing length of exogenous DNA inserts.
The IVC donor system enhances the efficiency of precise DNA insertion compared to standard circular donors.
Integration rates for 5.8, 7.5, and 11.1 kb fragments increased significantly, from 29.6% to 67.8%, 15.0% to 72.0%, and 12.1% to 65.6%, respectively.
The IVC donor also showed improved efficiency for a 7.4 kb fragment at an alternative target site.
This approach may be useful for advancing research and applications in synthetic biology and crop improvement.

Simplified

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.

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