多种Cas12a蛋白普适性的高性能低成本荧光检测缓冲体系优化研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1073

摘要/Abstract

摘要：

目的现有CRISPR/Cas12a检测体系多沿用限制性内切酶的通用反应缓冲液，但该类缓冲体系未充分考虑其在Cas12a荧光信号释放中的适配性，限制了检测灵敏度并增加了应用成本。为此，本研究旨在构建一种高适配性、低成本且适用于多种Cas12a蛋白的荧光优化反应体系，以显著提升核酸检测性能。方法通过荧光检测器定量分析与可视化检测，系统评估pH值（7.3-7.9，25 ℃）、Tris-HCl（5-50 mmol/L）、Ca²⁺（0.1-1 mmol/L）及Mg²⁺（10-30 mmol/L）对Cas12a荧光信号的影响。在此基础上，构建不含抗氧化剂及蛋白稳定剂的缓冲液CasRB，并与NEB系列商品缓冲液进行性能比较，同时验证其在FnCas12a、AsCas12a和LbCas12a三种蛋白体系中的通用性。结果优化后的CasRB（10 mmol/L Tris-HCl、0.1 mmol/L CaCl₂、20 mmol/L MgCl₂，pH 7.9，25 ℃）在不含高价组分（DTT和蛋白稳定剂）的条件下，与NEB系列商品缓冲液相比，成本降低超过99.9%；荧光信噪比提升10倍以上，显著增强裸眼判读效果，且表现出良好的跨菌源Cas12a蛋白适用性。结论本研究开发的CasRB缓冲液兼具成本优势和性能优势，解决了通用缓冲液在Cas12a荧光检测系统适配性不足的问题。

关键词: CRISPR/Cas12a, 缓冲液优化, 荧光检测, 核酸检测, 酶活性

Abstract:

Objective Current CRISPR/Cas12a nucleic acid detection systems commonly use conventional restriction enzyme buffers, which are not specifically optimized for Cas12a-mediated fluorescence activation. This limitation reduces detection sensitivity and increases system cost. The study aimed to construct a broadly compatible, cost-effective, and fluorescence-optimized reaction system for multiple Cas12a proteins to improve nucleic acid detection performance. Methods Fluorescence quantification using a fluorescence detector and visual readout were employed to evaluate systematically the effects of pH (7.3–7.9, 25 ℃), Tris-HCl concentration (5–50 mmol/L), calcium ion (Ca²⁺, 0.1–1 mmol/L), and magnesium ion (Mg²⁺, 10–30 mmol/L) on Cas12a fluorescence signal. Based on these results, a simplified reaction buffer (CasRB), free of antioxidants and protein stabilizers, was developed. CasRB performance was compared with commercial NEB buffers, and its compatibility was tested in three Cas12a orthologs: Francisella novicida Cas12a (FnCas12a), Acidaminococcus sp. Cas12a (AsCas12a), and Lachnospiraceae bacterium Cas12a (LbCas12a). Results The optimized CasRB reduced buffer cost by over 99.9% compared with commercial buffers by eliminating high-cost components such as dithiothreitol (DTT) and protein stabilizers. Fluorescence signal-to-noise ratio increased more than tenfold, significantly enhancing naked-eye visualization. CasRB showed strong cross-ortholog compatibility, providing comparable fluorescence performance in FnCas12a, AsCas12a, and LbCas12a systems. Conclusion Systematic optimization of reaction conditions produced a CasRB buffer that combined cost reduction and enhanced fluorescence sensitivity. The buffer addressed the compatibility limitations of conventional buffers in Cas12a-based nucleic acid detection systems, offering a versatile platform for multiple Cas12a proteins.

Key words: CRISPR/Cas12a, buffer optimization, fluorescence detection, nucleic acid detection, enzyme activity

李雅琦, 孙萌, 李秀丽, 魏静娜, 赵琳琳, 赵云平, 刘征辉, 苏蘩. 多种Cas12a蛋白普适性的高性能低成本荧光检测缓冲体系优化研究[J]. 生物技术通报, 2026, 42(4): 83-91.

LI Ya-qi, SUN Meng, LI Xiu-li, WEI Jing-na, ZHAO Lin-lin, ZHAO Yun-ping, LIU Zheng-hui, SU Fan. Optimization of a High-performance and Low-cost Fluorescence Detection Buffer with Broad Compatibility across Cas12a Orthologs[J]. Biotechnology Bulletin, 2026, 42(4): 83-91.

图/表 6

表1 Cas12a体系所用dsDNA激活片段、crRNA序列及引物信息

Table 1 dsDNA activator fragments, crRNA sequences, and primer information used in the Cas12a system

名称 Name	序列 Sequences（5'-3'）
dsDNA activator	CTCGCGAGTCCCAACACCAAGCTGGGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTT *GCTGCGTTCTTCATCGATGC* CAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCA
crRNA-F	TAATACGACTCACTATAGGGAATTTCTACTGTTGTAGAT
crRNA-R	GCATCGATGAAGAACGCAGCATCTACAACAGTAGAAATT
crRNA	GGGAAUUUCUACUGUUGUAGAUGCUGCGUUCUUCAUCGAUGC

图1 Cas12a蛋白的表达纯化及体外酶切活性分析A：Cas12a重组蛋白的SDS-PAGE分析；B：三种Cas12a蛋白的体外DNA酶切活性检测；上图：琼脂糖凝胶电泳分析crRNA引导下各Cas12a蛋白对质粒DNA底物的切割情况。下图：对质粒靶标的灰度定量分析结果（n=3），数据以均值±标准误表示。

Fig. 1 Expression, purification, and in vitro cleavage activity analysis of Cas12a proteinsA: SDS-PAGE analysis of recombinant Cas12a proteins; B: In vitro DNA cleavage assays of three Cas12a proteins; Top: Agarose gel electrophoresis showing crRNA-guided cleavage of plasmid DNA substrates by each Cas12a protein. Bottom: Quantitative grayscale analysis of plasmid targets (n=3). Data are presented as mean ± SEM.

图2 关键反应条件对CRISPR/Cas12a荧光生物传感器性能的影响A：pH值的影响；B：Tris-HCl浓度的影响；C：Ca²⁺浓度的影响；D：Mg²⁺浓度的影响。每个条件均展示：荧光信号实时检测曲线和信噪比统计分析（n=3，数据以均值±标准误表示；显著性差异采用GraphPad Prism 7软件进行Kruskal-Wallis检验并结合Dunn’s多重比较检验分析，以不同字母标注，P<0.05），以及470 nm蓝光照射下的肉眼观测结果。实验中设置阳性样本（+，含质粒靶标）与阴性对照（-，不含质粒靶标），用于评估体系的有效激活信号与背景信号。下同

Fig. 2 Effect of key reaction conditions for the CRISPR/Cas12a fluorescence biosensor performanceA: Effect of pH; B: Effect of Tris-HCl concentration; C: Effect of Ca²⁺ concentration; D: Effect of Mg²⁺ concentration. Each condition shows real-time fluorescence detection curves and signal-to-noise ratio analysis (n=3, data presented as mean ± SEM; significant differences determined using GraphPad Prism 7 with Kruskal-Wallis test followed by Dunn’s multiple comparisons test, indicated by different letters, P<0.05), as well as visual observation under 470 nm blue light illumination. Positive samples (+, with plasmid target) and negative controls (-, without plasmid target) were included to evaluate effective signal activation and background fluorescence. The same below

图3 CasRB与商品化缓冲液在CRISPR/Cas12a荧光检测体系中的性能对比A：荧光信号实时检测曲线；B：470 nm蓝光照射下的肉眼观测结果；C：荧光信号信噪比

Fig. 3 Performance comparison between CasRB and commercial buffers in the CRISPR/Cas12a fluorescence detection systemA: Real-time fluorescence detection curves; B: Visual observation under 470 nm blue light illumi nation; C: Signal-to-noise ratio of fluorescence detection

表2 CasRB与商品化缓冲液在60 min终点的信噪比（SNR）与成本对比

Table 2 Comparison of signal-to-noise ratio (SNR) and estimated cost between CasRB and commercial buffers at the 60 min endpoint

缓冲液体系Buffer	信噪比（均值±标准差） Signal-to-noise ratio （Mean ± SD）	相对信噪比 Relative SNR （CasRB=1）	单反应成本估算（分） Estimated cost per reaction （CNY cent）	相对成本 Relative cost （CasRB=1）
CasRB	1 672.8±110.9	1.00	0.015^a	1
NEBuffer 2	253.3±43.6	0.15	24.54^b	1 636
NEBuffer 2.1	525.4±82.8	0.31	24.54^b	1 636
NEBuffer 3.1	122.0±11.4	0.07	24.54^b	1 636
NEBuffer CutSmart	149.9±18.2	0.09	24.54^b	1 636

图4 CasRB对不同Cas12a蛋白的适用性评价A：荧光信号实时检测曲线；B：反应20分钟时荧光信号信噪比。

Fig. 4 Evaluation of the applicability of CasRB to different Cas12a enzymesA: Real-time fluorescence detection curves; B: Signal-to-noise ratio of fluorescence detection at 20 min.

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