Unleashing high trans-substrate cleavage kinetics of Cas12a for nucleic acid diagnostics

IF 13.1 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research Pub Date : 2025-07-26 DOI:10.1093/nar/gkaf712

Eric A Nalefski, Samantha Hedley, Karunya Rajaraman, Remy M Kooistra, Ishira Parikh, Selma Sinan, Ilya J Finklestein, Damian Madan

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引用次数: 0

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid diagnostics enable rapid, sensitive pathogen detection. Cas12a is frequently used in these assays because target-activated trans cleavage of a reporter molecule generates an easily detectable signal. However, variable activity across assays suggests that the catalytic potential of Cas12a has been limited via unknown mechanisms. Here, we show that Cas12a trans-nuclease activity is auto-inhibited by long PAM-proximal DNA (>120 bp) following cis-cleavage of targets. Short targets (<100 bp), optimized trans cleavage substrates, and low salt buffers unleash high catalytic efficiency (≈108 M−1 s−1) and turnover (≈1 s−1) across Cas12a orthologs. Pooling multiple Cas12a ribonucleoproteins (RNPs) targeting clustered protospacers overcomes cis-cleavage auto-inhibition, further boosting sensitivity. Optimized CRISPR RNA pools enable sub-femtomolar sensitivity for target detection without any pre-amplification. This mechanistic insight and mitigation strategy broaden the application of CRISPR–Cas enzymes for nucleic acid diagnostics.

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释放Cas12a高跨底物裂解动力学用于核酸诊断

基于CRISPR（聚集规律间隔短回文重复序列）的核酸诊断能够快速、敏感地检测病原体。Cas12a经常用于这些检测，因为靶激活的报告分子的反式切割产生易于检测的信号。然而，不同测试的不同活性表明，Cas12a的催化潜力受到未知机制的限制。在这里，我们发现Cas12a反式核酸酶活性在靶标顺式切割后被长pam -近端DNA （>120 bp）自动抑制。短靶点（<100 bp）、优化的反式裂解底物和低盐缓冲液释放出高催化效率（≈108 M−1 s−1）和Cas12a同源物的周转率（≈1 s−1）。汇集多个Cas12a核糖核蛋白（RNPs）靶向簇状原间隔物克服了顺式切割的自抑制，进一步提高了灵敏度。优化的CRISPR RNA池无需任何预扩增即可实现亚飞摩尔灵敏度的目标检测。这种机制的洞察力和缓解策略拓宽了CRISPR-Cas酶在核酸诊断中的应用。

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来源期刊

Nucleic Acids Research 生物-生化与分子生物学

CiteScore

27.10

自引率

4.70%

发文量

1057

审稿时长

2 months

期刊介绍： Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.