摘要
Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.
基金
supported by grants from the National Key Research and Development Program of China(2022YFF1002802)
the National Natural Science Foundation of China(32170410)
the Science and Technology Innovation Young Talent Team of Shanxi Province(202204051001019).
