Element coding based accurate evaluation of CRISPR/Cas9 initial cleavage,Chemical Science

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Element coding based accurate evaluation of CRISPR/Cas9 initial cleavage
Chemical Science ( IF 7.6 ) Pub Date : 2021-09-07 , DOI: 10.1039/d1sc03599a
Jianyu Hu ₁ , Rui Liu ₂ , Jing Zhou ₁ , Yi Lv _{1,

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Affiliation

As a powerful gene editing tool, the kinetic mechanism of CRISPR/Cas9 has been the focus for its further application. Initial cleavage events as the first domino followed by nuclease end trimming significantly affect the final on-target rate. Here we propose EC-CRISPR, element coding CRISPR, an accurate evaluation platform for initial cleavage that directly characterizes the cleavage efficiency and breaking sites. We benchmarked the influence of 19 single mismatch and 3 multiple mismatch positions of DNA-sgRNA on initial cleavage, as well as various reaction conditions. Results from EC-CRISPR demonstrate that the PAM-distal single mismatch is relatively acceptable compared to the proximal one. And multiple mismatches will not only affect the cleavage efficiency, but also generate more non-site #3 cleavage. Through in-depth research of kinetic behavior, we uncovered an abnormally higher non-#3 proportion at the initial stage of cleavage by using EC-CRISPR. Together, our results provided insights into cleavage efficiency and breaking sites, demonstrating that EC-CRISPR as a novel quantitative platform for initial cleavage enables accurate comparison of efficiencies and specificities among multiple CRISPR/Cas enzymes.

中文翻译：

基于元素编码的 CRISPR/Cas9 初始切割准确评估

作为一种强大的基因编辑工具，CRISPR/Cas9的动力学机制一直是其进一步应用的重点。最初的切割事件作为第一个多米诺骨牌，然后是核酸酶末端修整，显着影响最终的目标率。在这里，我们提出了 EC-CRISPR，元件编码 CRISPR，这是一个准确的初始切割评估平台，可直接表征切割效率和断裂位点。我们对 DNA-sgRNA 的 19 个单一错配和 3 个多重错配位置对初始切割以及各种反应条件的影响进行了基准测试。EC-CRISPR 的结果表明，与近端相比，PAM 远端单个错配相对可以接受。并且多次错配不仅会影响切割效率，还会产生更多的非位点#3 切割。通过对动力学行为的深入研究，我们通过使用 EC-CRISPR 在切割的初始阶段发现了异常较高的非 #3 比例。总之，我们的结果提供了对切割效率和断裂位点的见解，表明 EC-CRISPR 作为初始切割的新型定量平台能够准确比较多种 CRISPR/Cas 酶之间的效率和特异性。

更新日期：2021-09-22

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