Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called “hotspots.” In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.

中文翻译：

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基于 CRISPR-Cas12a 的体外 crRNA 种子区域以外的癌症相关 TP53 热点突变检测

具有成本效益和时间效率的致癌突变检测支持改善症状前癌症诊断和治疗后疾病监测。成簇规律间隔的短回文重复序列 (CRISPR)-Cas12a 是一种 RNA 引导的核酸内切酶，在依赖原型间隔区相邻基序 (PAM) 的顺式靶标 DNA 识别后，在反式中表现出不分青红皂白的 ssDNase 活性，可用于诊断。TP53 是癌症中突变最频繁的肿瘤抑制基因之一，在所谓的“热点”处显示反复出现的点突变。在这项研究中，我们优化了基于 Cas12a 的检测条件，用于体外检测六种TP53位于 Cas12a 种子区域外的 p.R273 密码子的热点突变，并评估了四种商业 Cas12a 变体的特异性。我们发现非工程 LbCas12a 明显优于其他测试的核酸酶，特别是在区分合成 DNA、模拟无细胞 DNA 和组织活检中的突变体 p.R273 密码子方面，尽管相应突变的 PAM 远端定位不理想。未来基于 Cas12a 的临床应用可能包括即时肿瘤分析、具有成本效益的突变筛查以及改进对个体癌症患者的监测。