Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR/Cas12a and Recombinase Polymerase Amplification

重组酶聚合酶扩增 化学 清脆的 聚合酶链反应 重组酶 聚合酶 计算生物学 单核苷酸多态性 遗传学 分子生物学 基因型 DNA 基因 生物化学 重组 生物
作者
Xingyue Wang,Ting Yang,Yunling Zhang,Zongyue Zeng,Qiang Wei,Pu Chen,Shuangshuang Yang,Yunfei Huang,Yongqi Zhang,Hongling Lu,Linhong Wu,Dijiao Tang,Ping Yang,Xuechun Wang,Qing Liu,Fan Li,Chao Ling,Shifeng Huang
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:96 (44): 17567-17575 被引量:3
标识
DOI:10.1021/acs.analchem.4c03288
摘要

Conventional methods for detecting single nucleotide polymorphisms (SNPs) in clinical practice often require substantial time, labor, and specialized equipment, limiting their widespread application. To address this limitation, we refined our previous SNP detection method, IMAS-RPA [introducing an extra mismatched base adjacent to the single-base mutant site by recombinase polymerase amplification (RPA)], resulting in an updated version termed IMAS-RPAv2. We began by introducing a suboptimal protospacer adjacent motif (PAM) sequence, GTTG, into the double-stranded DNA (dsDNA) products using either RPA or reverse transcription RPA. This modification decreased the efficiency with which CRISPR RNA (crRNA) recognizes the PAM and locally unwinds the dsDNA to form an R loop. After a delay, the R loop forms. However, due to the intentional incorporation of a mismatched base on the crRNA relative to the wild-type double-stranded DNA (WT-dsDNA), a continuous two-base mismatch is established between the crRNA and WT-dsDNA. Consequently, WT-dsDNA does not activate CRISPR/Cas12a's cleavage activity within a short time, while variant-type dsDNA continues to activate CRISPR/Cas12a and produce a robust fluorescence signal. This improvement significantly enhances the SNP discrimination sensitivity, allowing for detection at the single-copy level. Results were observed using both a conventional microplate reader and a specially designed portable device created through 3D printing. This device allows a direct fluorescence observation without the need for additional equipment. Consequently, the entire detection process becomes independent of large-scale equipment. This greatly expands its range of applications and offers promising prospects for clinical use.
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