Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection
Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection
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DOI：
10.1038/s41589-022-01135-y
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发表时间：
2022-09-22
影响因子：
14.8
通讯作者：
Gao, Xue
中科院分区：
文献类型：
--
作者：
Yang, Jie;Song, Yang;Gao, Xue
成簇规则间隔短回文重复序列 (CRISPR)-CRISPR 相关蛋白 13 (Cas13) 利用其特有的附带活性，已被迅速开发用于基于核酸的诊断。尽管最近在优化用于检测核酸的 Cas13 系统方面取得了进展，但设计具有增强的附带活性的 Cas13 蛋白一直具有挑战性，主要是因为其复杂的结构动力学。在这里，我们成功地采用了一种新的策略来改造 Leptotrichia wadei (Lwa)Cas13a，将不同的 RNA 结合结构域插入到其高等真核生物和原核生物核苷酸结合结构域内的独特活性位点近端环中。两种 LwaCas13a 变体在各种缓冲条件下表现出比野生型更强的附带活性和更高的敏感性。通过与电化学方法相结合，我们的变体从非活性病毒和未提取的临床样本中检测到阿摩尔浓度的 SARS-CoV-2 基因组，无需目标预扩增。我们的工程化 LwaCasl3a 酶具有增强的附带活性，可集成到其他基于 Cas13a 的平台中，以实现核酸的超灵敏检测。
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCasl3a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCasl3a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.