No abstract

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering primeediting guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP, and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A, and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2~10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated an alternative strategy for enhancing prime editing.

Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP, and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A, and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2~10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated a new strategy for more efficient prime editing and provide a platform for further directed evolution of PEs.