反式激活crRNA
单核苷酸多态性
清脆的
核糖核酸
计算生物学
生物
基因分型
遗传学
Cas9
基因
基因型
作者
Yuqing Ke,Shiyi Huang,Behafarid Ghalandari,Sijie Li,Antony R. Warden,Jingqi Dang,Lin Woo Kang,Yu Zhang,Yunqing Wang,Yiqing Sun,Jinglin Wang,Daxiang Cui,Xiao Zhi,Yiyang Li
标识
DOI:10.1002/advs.202003611
摘要
Abstract 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.
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