Plant Prime Editors Enable Precise Gene Editing in Rice Cells

Abstract

Prime editing events revealed by next-generation sequencing (NGS). (D) Quantification of prime editing frequencies by PPE3b-V01 at five target sites. (E) Editing events revealed by NGS reads. (F) Schematics of the expression vectors of Plant Prime Editor 3 or 2-Version 2 (PPE3/2-V02). (G-I) Comparison of multiple PBS-RT pairs of different lengths for directing TCA insertion at the OsPDS target site, directing C to A base change at the OsDEP1 target site and directing TGA insertion at the OsDEP1 target site, respectively, by PPE3-V02. (J and K) Validation of prime editing outcomes by NGS at the OsPDS-pegR15, OsDEP1-pegR03, and OsDEP1-pegR10 target sites. (M) Comparison of PPE3-V02 and PPE2-V02 for precise editing at five target sites. (N) PPE2-V02 based prime editing events revealed by NGS for the OsPDS-sgRNA01 3T ins construct (for insertion of a T 3 nt downstream of the PBS). (O) PPE2-V02 based prime editing at another site with multiple PBS-RT pairs of different lengths. The experiments were done in rice protoplasts.Three biological replicates were used to assess the PPE3-V01 system (B-E), and two biological replicates were used to assess PPE3-V02 and PPE2-V02 systems (G-O). Error bars represent standard deviations of the biological replicates. For NGS-based genotyping data presentations (C, E, J, K, L, and N), the sequences (from top to bottom) include the wild-type (WT) sequence (protospacer underlined and PAM in bold), the expected prime editing outcome (Reference), confirmed precise prime editing events matching the expected prime editing outcome (PE_Ref), precise prime editing plus additional single nucleotide polymorphisms (e.g., PE_h01; h stands for haplotype) and deletions resulted from the NHEJ repair. The prime edited DNA nucleotides are highlighted in red.

Prime editing is achievable in the tetraploid potato, but needs improvement

“…Very recently, several reports have shown that prime editing can be applied to cereal plants Lin et al, 2020;Tang et al, 2020;Xu et al, 2020a;Xu et al, 2020b;Butt et al;Hua et al). All these studies showed that the efficiency of PPEs greatly varies between target sites and is sensitive to several parameters such as both the PBS and RT sequence.…”

Precision genome engineering in rice using prime editing system

“…In summary, the prime editing tool could precisely edit the transgenic reporter and endogenous gene in rice. During the preparation and review of our manuscript, several other prime editing systems in rice and wheat were reported (Li et al, 2020;Lin et al, 2020;Tang et al, 2020;Xu et al, 2020a;Xu et al, 2020b). Tang and colleagues presented data from rice protoplasts only and did not obtain any stable transgenic lines with editing events (Tang et al, 2020), whereas 1-3 rice endogenous genes were edited with relatively low efficiencies in transgenic plants in the other studies.…”

“…Tang and colleagues presented data from rice protoplasts only and did not obtain any stable transgenic lines with editing events (Tang et al, 2020), whereas 1-3 rice endogenous genes were edited with relatively low efficiencies in transgenic plants in the other studies. It is possible that the editing efficiency of the prime editing system can be improved by using other reverse transcriptases (Stamos et al, 2017) or modifying the transgene selection system (Li et al, 2019) to make the prime editors powerful tools for precision molecular breeding of crops.…”

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

“…Recently, a novel and universal precision genome-editing technology named prime editing was developed and tested in mammalian cells [1]. More recently, seven groups reported applications of prime editors (PE) in rice and wheat, and six groups achieved prime-edited rice lines [2][3][4][5][6][7][8]. Excluding the data based on enriching strategies or that are unsuitable for comparison, a total of 164 prime-edited rice lines transformed with 39 prime editors targeting 11 endogenous rice genes were generated in these previous reports [2,3,5,6,8].…”