生物技术通报2026，Vol.42Issue(4)：83-91,9.DOI:10.13560/j.cnki.biotech.bull.1985.2025-1073
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
Optimization of a High-performance and Low-cost Fluorescence Detection Buffer with Broad Compatibility across Cas12a Orthologs
摘要
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.[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.关键词
CRISPR/Cas12a/缓冲液优化/荧光检测/核酸检测/酶活性Key words
CRISPR/Cas12a/buffer optimization/fluorescence detection/nucleic acid detection/enzyme activity引用本文复制引用
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[J].生物技术通报,2026,42(4):83-91,9.基金项目
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) （32201252）
