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Abstract：
The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.
摘要：
sgRNA-DNA互补序列的长度是影响化脓性链球菌Cas9(SpCas9)及其变体的裂解活性的关键因素。详细的机制仍然未知。这里,基于体外裂解试验和碱基编辑分析，我们证明,减少该互补区域的长度可以赋予SpCas9和eSpCas9的切口酶活性(1.1)。我们还显示这些切口是在靶DNA链上产生的。这些特性鼓励我们开发一种双功能系统，该系统同时在不同的靶标处进行双链DNA切割和C到T碱基转化。该系统提供了一种用于在植物中实现性状堆叠的新颖工具。
