<br>酶动力学和检测器灵敏度决定无扩增 CRISPR-Cas12 和 CRISPR-Cas13 诊断的检测限,Analytical Chemistry

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酶动力学和检测器灵敏度决定无扩增 CRISPR-Cas12 和 CRISPR-Cas13 诊断的检测限
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-27 , DOI: 10.1021/acs.analchem.2c01670
Diego A Huyke ₁ , Ashwin Ramachandran ₂ , Vladimir I Bashkirov ₃ , Efthalia K Kotseroglou ₃ , Theofilos Kotseroglou ₃ , Juan G Santiago ₁

Affiliation

人们对 CRISPR-Cas12 和 CRISPR-Cas13 检测的兴趣不断增加，因为这些检测方案能够特异性识别核酸。这些方案的基本灵敏度限制（及其在无扩增测定中的适用性）由动力学速率控制。然而，人们对这些动力学速率仍然知之甚少，而且它们的报告也不一致。我们量化了几种酶（LbCas12a、AsCas12a、AapCas12b、LwaCas13a 和 LbuCas13a）的动力学参数及其相应的检测限 (LoD)。总的来说，我们对 14 种向导 RNA (gRNA) 和核酸靶标的酶动力学进行了量化，总共 50 组动力学速率参数和 25 个 LoD。我们通过将趋势和限制行为与 Michaelis-Menten反式裂解反应动力学模型进行比较，验证了我们测量的自我一致性。对于我们的测定条件，活化的 Cas12 和 Cas13 酶表现出 10 ⁵和 10 ⁶ M ^–1 s ^–1级之间的反式裂解催化效率。对于使用荧光报告分子（ssDNA 和 ssRNA）进行靶标检测的测定，当前测定条件下的动力学速率会导致皮摩尔范围内的无扩增 LoD。结果表明，成功检测目标需要至少 0.1% 的荧光报告分子裂解（通过激活的 CRISPR 酶）。需要这部分被切割的报告基因来区分信号与背景，并且我们假设所需的部分在很大程度上独立于检测方法（例如，终点与反应速度）和检测器灵敏度。我们的结果证明了动力学速率和背景信号限制 LoD 的基本性质，从而突出了 CRISPR 诊断这一新兴领域的改进领域。

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Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics

Interest in CRISPR-Cas12 and CRISPR-Cas13 detection continues to increase as these detection schemes enable the specific recognition of nucleic acids. The fundamental sensitivity limits of these schemes (and their applicability in amplification-free assays) are governed by kinetic rates. However, these kinetic rates remain poorly understood, and their reporting has been inconsistent. We quantify kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a, and LbuCas13a) and their corresponding limits of detection (LoD). Collectively, we present quantification of enzyme kinetics for 14 guide RNAs (gRNAs) and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. We validate the self-consistency of our measurements by comparing trends and limiting behaviors with a Michaelis–Menten trans-cleavage reaction kinetics model. For our assay conditions, activated Cas12 and Cas13 enzymes exhibit trans-cleavage catalytic efficiencies between order 10⁵ and 10⁶ M^–1 s^–1. For assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, the kinetic rates at the current assay conditions result in an amplification-free LoD in the picomolar range. The results suggest that successful detection of target requires cleavage (by an activated CRISPR enzyme) of the order of at least 0.1% of the fluorescent reporter molecules. This fraction of reporters cleaved is required to differentiate the signal from the background, and we hypothesize that this required fraction is largely independent of the detection method (e.g., endpoint vs reaction velocity) and detector sensitivity. Our results demonstrate the fundamental nature by which kinetic rates and background signal limit LoDs and thus highlight areas of improvement for the emerging field of CRISPR diagnostics.

更新日期：2022-06-27

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