Hairpin‐Spacer crRNA‐Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification,Advanced Science

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Hairpin‐Spacer crRNA‐Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification
Advanced Science ( IF 14.3 ) Pub Date : 2021-01-31 , DOI: 10.1002/advs.202003611
Yuqing Ke ₁ , Shiyi Huang ₁ , Behafarid Ghalandari ₁ , Sijie Li ₁ , Antony R Warden ₁ , Jingqi Dang ₁ , Lin Kang ₂ , Yu Zhang ₁ , Yunqing Wang ₁ , Yiqing Sun ₁ , Jinglin Wang ₁ , Daxiang Cui ₃ , Xiao Zhi ₁ , Xianting Ding ₁

Affiliation

The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs‐CRISPR). Gibbs free energy analysis suggests that the hairpin‐spacer crRNAs (hs‐crRNAs) suppress Cas13a's affinity to off‐target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high‐specificity of hs‐CRISPR/Cas13a system. Compared to ordinary crRNA, hs‐crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs‐crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.

中文翻译：

发夹间隔区 crRNA 增强的 CRISPR/Cas13a 系统提高单核苷酸多态性 (SNP) 鉴定的特异性

Cas13a系统在RNA干扰和分子诊断领域具有巨大潜力。然而，缺乏crRNA设计指南阻碍了Cas13a系统在RNA编辑和单核苷酸多态性鉴定中的实际应用。本研究假设带有发夹间隔区的 crRNA 提高了 CRISPR/Cas13a 系统（称为 hs-CRISPR）的特异性。吉布斯自由能分析表明发夹间隔 crRNA (hs-crRNA) 抑制 Cas13a 对脱靶 RNA 的亲和力。建立乙型肝炎病毒DNA基因分型平台，进一步验证hs-CRISPR/Cas13a系统的高特异性。与普通crRNA相比，hs-crRNA在不牺牲CRISPR/Cas13a系统灵敏度的情况下将特异性提高了三倍。此外，通过理论模拟阐明了Cas13a/hs-crRNA/靶RNA组成的机制。这项工作建立在对 Cas13a 激活的基本理解之上，并为 CRISPR/Cas13a 系统的 crRNA 的合理设计提供了重大改进。

更新日期：2021-03-17

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