Vu Tien Van, Nguyen Ngan Thi, Kim Jihae, Das Swati, Lee Jinsu, Kim Jae-Yean
Division of Applied Life Science (BK21 FOUR Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea.
National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Km 02, Pham Van Dong Road, Co Nhue 1, Bac Tu Liem, Hanoi 11917, Vietnam.
Biodes Res. 2022 Dec 15;2022:0001. doi: 10.34133/bdr.0001. eCollection 2022.
Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA (gRNA) has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs (pegRNAs) negatively affected the cleaving activity of the Cas9 nuclease. The self-complementarity between the primer binding sequences (PBSs) and spacer sequence might pose risks to the activity of the Cas9 complex. However, modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein (SlMBP21), alcobaca (SlALC), and acetolactate synthase 1 (SlALS1) loci. Our data show challenges of the PE approach in tomato, indicating that a further improvement of the PE system for successful applications is demanded, such as the use of improved expression systems for enriching active PE complexes.
精准基因组编辑对于作物改良具有很高的需求。最近出现的CRISPR/Cas技术在精准植物基因组工程中具有巨大的潜在应用价值。一种将逆转录酶（RT）与Cas9切口酶以及“引导”延伸向导RNA（gRNA）相结合的碱基编辑（PE）方法，已在哺乳动物细胞和几种植物物种中显示出对基因组进行精确修饰的高频率。然而，PE方法在双子叶植物中的应用仍然有限且效率低下。我们设计并测试了碱基编辑器，用于在瞬时检测中对合成序列进行精准编辑，并通过稳定转化对番茄中10个位点的理想等位基因进行编辑。我们通过靶向深度测序获得的数据还显示，在烟草和番茄系统中PE效率都很低。对PE组件活性的进一步评估发现，RT与Cas9的融合以及PE gRNAs（pegRNAs）的结构对Cas9核酸酶的切割活性产生了负面影响。引物结合序列（PBSs）与间隔序列之间的自我互补性可能会对Cas9复合物的活性构成风险。然而，通过缩短pegRNA序列或在PBSs中引入错配以降低其解链温度，并没有提高MADS盒蛋白（SlMBP21）、醇脱氢酶（SlALC）和乙酰乳酸合酶1（SlALS1）位点的PE效率。我们的数据显示了PE方法在番茄中的挑战，表明需要进一步改进PE系统以实现成功应用，例如使用改进的表达系统来富集活性PE复合物。
