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OA
Optimization of a High-performance and Low-cost Fluorescence Detection Buffer with Broad Compatibility across Cas12a Orthologs
[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.
[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.[Method]Fluorescence quantification using a fluorescence detector and visual readout were employed to systematically evaluate 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).[Result]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 produce a CasRB buffer that combined cost reduction and enhanced fluorescence sensitivity.The buffer addresses the compatibility limitations of conventional buffers in Cas12a-based nucleic acid detection systems,offering a versatile platform for multiple Cas12a proteins.
李雅琦;孙萌;李秀丽;魏静娜;赵琳琳;赵云平;刘征辉;苏蘩
天津市农业科学院,天津 300381||北京林业大学林学院,北京 100083天津市农业科学院,天津 300381天津市农业科学院,天津 300381天津市农业科学院,天津 300381天津市农业科学院,天津 300381天津市农业科学院,天津 300381天津市农业科学院,天津 300381天津市农业科学院,天津 300381
CRISPR/Cas12a缓冲液优化荧光检测核酸检测酶活性
CRISPR/Cas12abuffer optimizationfluorescence detectionnucleic acid detectionenzyme activity
《生物技术通报》 2026 (4)
83-91,9
国家自然科学基金项目(32201252),天津市农业科学院青年科研人员创新研究与实验项目(2022008),天津市农业质量标准与检测技术研究所科研创新基金项目(ZBS-2005)
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