DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
BrisSynBio, Life Sciences Building, Tyndall Avenue, University of Bristol, Bristol BS8 1TQ, UK.
Nucleic Acids Res. 2020 Jul 9;48(12):6811-6823. doi: 10.1093/nar/gkaa477.
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 used in vitro ensemble 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 of in vitro transcribed 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.
在利用 CRISPR-Cas 时，一个关键目标是通过 gRNA 工程引入额外的功能，范围从提高靶标结合效率的单个核苷酸变化到包含更大功能的 RNA 适体或核糖核蛋白 (RNP)。Cas9-gRNA 相互作用对于复杂组装至关重要，但 gRNA 的几个不同区域可以进行修饰。我们使用体外集合和单分子测定来评估 gRNA 结构改变对 RNP 复合物形成、R 环动力学和内切酶活性的影响。我们的结果表明，RNP 形成不受任何修饰的影响。R 环形成和 DNA 切割活性也基本上不受上茎、第一发夹和 3' 端修饰的影响。相比之下，我们发现相对于单个核苷酸添加，仅添加两个或三个核苷酸的 5' 可减少 RuvC 结构域的 R 环形成和切割活性。这种修饰是体外转录的 gRNA 的常见副产物。我们还观察到，将 20nt RNA 发夹添加到 gRNA 的 5' 端仍支持 RNP 形成，但产生了不能激活 DNA 切割的稳定的~9bp R 环。对这些观察结果的考虑将有助于成功的 gRNA 设计。
