5' modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage
5' modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage
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CRISPR Cas9 gRNA 的 5 修饰可以改变 R 环的动态和大小并抑制 DNA 切割
DOI：
10.1101/2020.04.09.033399
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Mullally G
中科院分区：
文献类型：
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作者：
Mullally G
利用CRISPR-CAS的一个关键目标是通过gRNA工程引入额外的功能，从增加靶标结合效率的单个核苷酸变化到包含更大的功能RNA适配子或核糖核蛋白(RNP)。Cas9-gRNA相互作用对于复杂的组装至关重要，但gRNA的几个不同区域可以进行修饰。我们使用玻璃体系综和单分子分析来评估gRNA结构变化对RNP复合体形成、R环动力学和核酸内切酶活性的影响。我们的结果表明，我们的任何修饰都不影响RNP的形成。R环的形成和DNA切割活性也基本上不受上茎、第一发夹和3‘端的修饰。相反，我们发现只有两个或三个核苷酸的5‘加成比单核苷酸加成减少了RuvC结构域的R环形成和切割活性。这样的修饰是体外转录gRNA的常见副产品。我们还观察到，在gRNA5‘端添加20ntRNA发夹仍然支持RNP的形成，但产生了稳定的∼9bP R环，不能激活DNA裂解。考虑这些观察结果将有助于成功的gRNA设计。
A key aim in exploiting CRISPR–Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We usedin vitroensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product ofin vitrotranscribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.