Single-molecule assay guided crRNA optimization enhances specific microRNA detection by CRISPR-Cas12a
文献类型： 外文期刊
作者： Chen, Kaizhao 1 ; Sun, Wenjun 3 ; Zhong, Mingtian 1 ; Xie, Jiaqi 1 ; Huo, Yongkun 1 ; Lu, Xiuqi 3 ; Chen, Ziting 3 ; Sun, Bo 3 ; Huang, Xingxu 3 ; Wang, Xinjie 5 ; Liu, Ming 6 ; Ma, Xiaodong 1 ; Ma, Peixiang 7 ;
作者机构： 1.Minist Educ, Key Lab Brain, Cognit & Educ Sci, Beijing, Peoples R China
2.South China Normal Univ, Inst Brain Res & Rehabil, Guangzhou 510631, Peoples R China
3.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China
4.Zhejiang Univ, Affiliated Hosp 1, Key Lab Pancreat Dis Zhejiang Prov, Sch Med, Hangzhou 310003, Peoples R China
5.Chinese Acad Agr Sci, Genom Inst Shenzhen, Guangdong Lab Lingnan Modern Agr, Shenzhen Branch,Genome Anal Lab,Minist Agr & Rural, Shenzhen 518120, Peoples R China
6.Guangzhou Med Univ, Affiliated Hosp 1, Guangzhou Inst Resp Hlth, Natl Clin Res Ctr Resp Dis,State Key Lab Resp Dis, Guangzhou 510120, Peoples R China
7.Shanghai Jiao Tong Univ, Shanghai Peoples Hosp 9, Dept Orthoped Surg, Shanghai Key Lab Orthoped Implants,Sch Med, Shanghai 200025, Peoples R China
关键词： MicroRNA; CRISPR; Cas12a; Stem-loop; Detection
期刊名称：SENSORS AND ACTUATORS B-CHEMICAL （ 影响因子：8.4； 五年影响因子：7.2 ）
ISSN：
年卷期： 2024 年 406 卷
页码：
收录情况： SCI
摘要： CRISPR-Cas12a is a promising tool for nucleic acid detection. However, due to the protein flexibility, Cas12a tolerates mismatches, which limits its specificity. In this study, the single-molecule assay revealed that the length of crRNA regulates the association kinetics between crRNA/Cas12a complex and target DNA. Short-crRNA/ Cas12 associates the target dsDNA 2-fold faster than the single-nucleotide mismatched dsDNA, whereas the long-crRNA/Cas12a binds to the fully matched and single-nucleotide mismatched DNA targets with similar rates. These findings are further corroborated by electrophoretic mobility shift assay (EMSA) and double-stranded DNA (dsDNA) cleavage results. Inspired by these findings, we established stem-loop amplification conjugated short crRNA CRISPR-Cas12a (SlashCas12a) detection. In this approach, stem-loop-mediated microRNA reverse transcription was harnessed to enhance the amplification efficiency of the short single-stranded RNA. The shortcrRNA Cas12a specifically distinguishes the single mismatched miRNA homologs, i.e. let-7a family members. The introduction of PAM containing stem-loop can break through the limitation of PAM for Cas12a and efficiently amplify the miRNAs. Multiple miRNAs, including miR-122b, miR-21, and let-7a, can be efficiently detected, and the limit of detection is up to 7.8 aM. Furthermore, the distinct expression of miR-21 and let-7a can be detected in the lung adenocarcinoma and breast samples using this method. These results demonstrate the appropriate crRNA engineering will extend the application of Cas12a in the specific molecule diagnosis.
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