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发夹-间隔区crRNA增强的CRISPR/Cas13a系统促进单核苷酸多态性（SNP）鉴定的特异性。

Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification.

作者信息

Ke Yuqing, Huang Shiyi, Ghalandari Behafarid, Li Sijie, Warden Antony R, Dang Jingqi, Kang Lin, Zhang Yu, Wang Yunqing, Sun Yiqing, Wang Jinglin, Cui Daxiang, Zhi Xiao, Ding Xianting

机构信息

State Key Laboratory of Oncogenes and Related Genes Institute for Personalized Medicine School of Biomedical Engineering Shanghai Jiao Tong University Shanghai 200030 China.

State Key Laboratory of Pathogen and Biosecurity Institute of Microbiology and Epidemiology Beijing 100071 China.

出版信息

Adv Sci (Weinh). 2021 Jan 31;8(6):2003611. doi: 10.1002/advs.202003611. eCollection 2021 Mar.

DOI:10.1002/advs.202003611
PMID:33747742
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7967054/
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.

摘要

Cas13a系统在RNA干扰和分子诊断领域具有巨大潜力。然而，缺乏针对crRNA设计的指导原则阻碍了Cas13a系统在RNA编辑和单核苷酸多态性鉴定中的实际应用。本研究提出，带有发夹间隔区的crRNA可提高CRISPR/Cas13a系统的特异性（称为hs-CRISPR）。吉布斯自由能分析表明，发夹间隔区crRNA（hs-crRNAs）可抑制Cas13a对脱靶RNA的亲和力。建立了一个乙型肝炎病毒DNA基因分型平台，以进一步验证hs-CRISPR/Cas13a系统的高特异性。与普通crRNA相比，hs-crRNAs在不牺牲CRISPR/Cas13a系统敏感性的情况下，将特异性提高了三倍。此外，通过理论模拟阐明了Cas13a/hs-crRNA/靶标RNA组成的机制。这项工作基于对Cas13a激活的基本理解，为CRISPR/Cas13a系统的crRNA合理设计提供了显著改进。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/7c4c5ea2e6d8/ADVS-8-2003611-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/0b4005ef2fc8/ADVS-8-2003611-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/985fe5a50acc/ADVS-8-2003611-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/aa251edbf743/ADVS-8-2003611-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/279c5c61342e/ADVS-8-2003611-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/7c4c5ea2e6d8/ADVS-8-2003611-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/0b4005ef2fc8/ADVS-8-2003611-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/985fe5a50acc/ADVS-8-2003611-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/aa251edbf743/ADVS-8-2003611-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/279c5c61342e/ADVS-8-2003611-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e9f5/7967054/7c4c5ea2e6d8/ADVS-8-2003611-g003.jpg

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