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.

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

No abstract

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely undetermined. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal different guide RNA (gRNA)-dependent off-target effects with different editors. De novo generated new gRNAs by SpRY editors lead to additional but not substantial off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. The preference of the TA motif by ABE8e is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scales and mechanisms of off-target and background mutations during PAM-relaxed genome editing in plants.

No abstract