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. 2021 Oct;39(10):1292-1299. doi: 10.1038/s41587-021-00891-x. Epub 2021 Apr 15.
Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome.
尽管主要编辑器 (PEs) 具有在治疗、农业和研究应用中促进精确基因组编辑的潜力，但它们的特异性尚未得到全面评估。为了在植物中进行系统评估，我们首先检查了 PEs 在植物细胞中的错配容忍度，发现编辑频率受引物结合位点和主要编辑指导 RNA (pegRNA) 间隔区中错配数量和位置的影响。在 179 个预测的脱靶位点评估 12 个 pegRNA 的活性，我们仅检测到低频率的脱靶编辑（0.00~0.23%）。对 29 株经 PE 处理的水稻的全基因组测序证实，PE 不会诱导全基因组 pegRNA 非依赖性脱靶单核苷酸变异或小插入/缺失。我们还表明，PE 中作为 Moloney 鼠白血病病毒逆转录酶的一部分的异位表达不会改变反转录转座子拷贝数或端粒结构，也不会导致 pegRNA 或信使 RNA 序列插入基因组。
