Prime editing enables precise genome modification of a Populus hybrid

This study is led by Professor Renying Zhuo (State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China). The authors used the 2×35S promoter to express a fusion protein of spCas9 nickase (nCas9) and engineered Moloney murine leukemia virus (MMLV), and the Arabidopsis thaliana AtU6 promoter to express an engineered prime editing (PE) guide RNA (epegRNA) and Nick gRNA, pioneering the establishment of the Prime Editor 3 (PE3) system in dicot poplar 84K (Populus alba × P. glandulosa). Three endogenous genes were selected as PE targets: PHYTOENE DESATURASE (PagPDS; Potri.014G148700), a homolog of Arabidopsis AtYUCCA4 (PagYUC4; Potri.006G248200), and SHORT ROOT (PagSHR; Potri.007G063300). To assess the editing capabilities of this method, three types of RT template were designed, including one single-base substitution, multiple-base substitutions, and small DNA fragment insertions/deletions.

To assess the feasibility of the PE3 system in 84K early on, the authors prioritized evaluating PE efficiencies in resistant callus. For single-base substitution, all three targets exhibited the desired edits with average efficiencies of 0.49% (PagPDS), 0.08% (PagYUC4), and 1.83% (PagSHR). For multiple-base substitutions, the PagPDS-g2 and PagYUC4-g2 targets were edited with average efficiencies of 3.82% and 3.62%. Desired small DNA fragment insertions/deletions were detected for the PagPDS-g3 and PagYUC4-g3 targets, with average efficiencies of 1.59% and 0.22%, respectively. To further validate the reliability of PE, the authors regenerated stable transgenic T₀ plants and used NGS to identify the editing types in all plants. The authors identified one desired edit (1/25, 4.0%) at the PagPDS-g1 target (single-base substitution) and five desired edits (5/32, 15.6%) at the PagPDS-g2 target (multiple-base substitutions). For PagYUC4 and PagSHR targets, desired edits were obtained at the PagSHR-g1 target (1/28, 3.6%; single-base substitution) and the PagYUC4-g2 target (8/36, 22.2%; multiple-base substitutions).

In summary, the authors successfully established a PE3 system in dicot poplar and obtained stable T₀ plants with the desired edits. Currently, the efficiency of the PE3 system in poplar is low and unstable, particularly for small-fragment insertions/deletions. In future, the PE3 system could be further optimized through methods effective in major crops, such as increasing epegRNA expression, enhancing MMLV activity, or implementing appropriate heat treatments.

See the article:

Prime editing enables precise genome modification of a Populus hybrid

https://link.springer.com/article/10.1007/s42994-024-00177-1