State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
Nat Biotechnol. 2022 Sep;40(9):1394-1402. doi: 10.1038/s41587-022-01254-w. Epub 2022 Mar 24.
Prime editing is a versatile genome-editing technology, but it suffers from low editing efficiency. In the present study, we introduce optimized prime editors with substantially improved editing efficiency. We engineered the Moloney-murine leukemia virus reverse transcriptase by removing its ribonuclease H domain and incorporated a viral nucleocapsid protein with nucleic acid chaperone activity. Each modification independently improved prime editing efficiency by ~1.8-3.4-fold in plant cells. When combined in our engineered plant prime editor (ePPE), the two modifications synergistically enhanced the efficiency of base substitutions, deletions and insertions at various endogenous sites by on average 5.8-fold compared with the original PPE in cell culture. No significant increase in byproducts or off-target editing was observed. We used the ePPE to generate rice plants tolerant to sulfonylurea and imidazolinone herbicides, observing an editing frequency of 11.3% compared with 2.1% using PPE. We also combined ePPE with the previously reported dual-prime editing guide (peg) RNAs and engineered pegRNAs to further increase efficiency.
先导编辑是一种多功能的基因组编辑技术，但编辑效率较低。在本研究中，我们引入了经过优化的先导编辑器，其编辑效率有了显著提高。我们通过去除 Moloney-鼠白血病病毒逆转录酶的核糖核酸酶 H 结构域，并加入具有核酸伴侣活性的病毒核衣壳蛋白，对其进行了工程改造。这两种修饰各自独立地将植物细胞中的先导编辑效率提高了约 1.8-3.4 倍。当这两种修饰结合到我们工程化的植物先导编辑器 (ePPE) 中时，与原始 PPE 相比，它们在细胞培养中的各种内源性位点的碱基替换、缺失和插入效率协同提高了 5.8 倍。没有观察到副产物或脱靶编辑的显著增加。我们使用 ePPE 生成了对磺酰脲和咪唑啉酮除草剂具有耐受性的水稻植株，与使用 PPE 相比，观察到的编辑频率为 11.3%。我们还将 ePPE 与之前报道的双先导编辑向导 (peg) RNA 结合，并对 pegRNA 进行了工程改造，以进一步提高效率。
