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High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems
ACS Sensors ( IF 8.2 ) Pub Date : 2023-11-27 , DOI: 10.1021/acssensors.3c02158 Yawen He 1 , Shengjie Shao 1 , Juhong Chen 1
ACS Sensors ( IF 8.2 ) Pub Date : 2023-11-27 , DOI: 10.1021/acssensors.3c02158 Yawen He 1 , Shengjie Shao 1 , Juhong Chen 1
Affiliation
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Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 103 copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.
中文翻译：
V 型 CRISPR 系统高保真度鉴定单核苷酸多态性
准确、灵敏的单核苷酸多态性（SNP）检测具有重要的临床意义，特别是在癌症诊断领域。凭借其高精度和可编程性，CRISPR 系统成为推进 SNP 识别的有前途的平台。在本研究中，我们比较了两种 V 型 CRISPR/Cas 系统（Cas12a 和 Cas14a），以鉴定与癌症相关的 SNP。通过表征它们对 BRAF 基因的错配耐受性来评估它们的识别性能。我们发现 CRISPR/Cas14a 系统在 SNP 检测方面表现出优于 CRISPR/Cas12a 系统的准确性和稳健性。此外，将阻断剂置换扩增（BDA）与CRISPR/Cas14a系统相结合，消除野生型（WT）的干扰，提高检测精度。在此策略中，我们能够以 0.1% 变异等位基因频率的灵敏度检测低至 10 3个拷贝的 BRAF V600E。此外，BDA辅助的CRISPR/Cas14a系统已应用于鉴定人结直肠癌细胞的BRAF突变，实现了0.5%变异等位基因频率的高灵敏度，与大多数市售产品相当甚至优于大多数市售产品。这项工作拓宽了 CRISPR 系统的范围，为精准医疗提供了一种有前途的方法。
更新日期：2023-11-27
中文翻译：
V 型 CRISPR 系统高保真度鉴定单核苷酸多态性
准确、灵敏的单核苷酸多态性（SNP）检测具有重要的临床意义，特别是在癌症诊断领域。凭借其高精度和可编程性，CRISPR 系统成为推进 SNP 识别的有前途的平台。在本研究中，我们比较了两种 V 型 CRISPR/Cas 系统（Cas12a 和 Cas14a），以鉴定与癌症相关的 SNP。通过表征它们对 BRAF 基因的错配耐受性来评估它们的识别性能。我们发现 CRISPR/Cas14a 系统在 SNP 检测方面表现出优于 CRISPR/Cas12a 系统的准确性和稳健性。此外，将阻断剂置换扩增（BDA）与CRISPR/Cas14a系统相结合，消除野生型（WT）的干扰，提高检测精度。在此策略中，我们能够以 0.1% 变异等位基因频率的灵敏度检测低至 10 3个拷贝的 BRAF V600E。此外，BDA辅助的CRISPR/Cas14a系统已应用于鉴定人结直肠癌细胞的BRAF突变，实现了0.5%变异等位基因频率的高灵敏度，与大多数市售产品相当甚至优于大多数市售产品。这项工作拓宽了 CRISPR 系统的范围，为精准医疗提供了一种有前途的方法。
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