Optimization of a High-performance and Low-cost Fluorescence Detection Buffer with Broad Compatibility across Cas12a Orthologs

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

Abstract

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

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.

Figures/Tables 6

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

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.

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

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

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

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.

References 31

[1]	Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity [J]. Science, 2018, 360(6387): 436-439.
[2]	Xin XL, Su J, Cui HR, et al. Recent advances in clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system-based biosensors [J]. Biosensors, 2025, 15(3): 155.
[3]	Pan LY, Jiang WW, Deng F, et al. Signal-amplification strategy and advances in electrochemistry-based CRISPR/Cas12 biosensing [J]. Chem Eng J, 2025, 508: 161110.
[4]	Swarts DC, Jinek M. Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a [J]. Mol Cell, 2019, 73(3): 589-600.e4.
[5]	Son H, Park J, Choi YH, et al. Exploring the dynamic nature of divalent metal ions involved in DNA cleavage by CRISPR-Cas12a [J]. Chem Commun, 2022, 58(12): 1978-1981.
[6]	Song Y, Xu YW, Wang RR, et al. CRISPR-Cas12a-based nanoparticle biosensor for detection of pathogenic bacteria in food [J]. Microchem J, 2024, 207: 111813.
[7]	Ma YJ, Wei HJ, Wang YX, et al. Efficient magnetic enrichment cascade single-step RPA-CRISPR/Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples [J]. J Hazard Mater, 2024, 465: 133494.
[8]	田道明, 周子萱, 杨迎澳, 等. CRISPR-Cas12a介导的适配体荧光传感器快速检测金黄色葡萄球菌 [J]. 中国医药科学, 2024(21): 139-143.
	Tian DM, Zhou ZX, Yang YA, et al. Rapid detection of Staphylococcus aureus by aptamer fluorescence sensor mediated by CRISPR-Cas12a [J]. China Medicine and Pharmacy, 2024(21): 139-143.
[9]	He W, Liao K, Li RX, et al. Development of a CRISPR/Cas12a-based fluorescent detection method of senecavirus A [J]. BMC Vet Res, 2024, 20(1): 258.
[10]	Cao ZZ, Li Z, Jin KK, et al. A rapid and visual detection method for porcine delta coronavirus with single-copy sensitivity based on the CRISPR/Cas12a assay [J]. J Integr Agric, 2025
[11]	热则古丽·艾科拜尔, 张亚平, 刘浩然, 等. 基于RAA-CRISPR/Cas12a建立牛病毒性腹泻病毒的可视化快速检测方法及其初步应用 [J]. 中国兽医科学, 2025(3): 321-329.
	Reze Guli A, Zhang YP, Liu HR, et al. Establishment of a visual rapid detection method for bovine viral diarrhea virus based on RAA-CRISPR/Cas12a and its preliminary application [J]. Chinese Veterinary Science, 2025(3): 321-329.
[12]	Shashank PR, Parker BM, Rananaware SR, et al. CRISPR-based diagnostics detects invasive insect pests [J]. Mol Ecol Resour, 2024, 24(1): e13881.
[13]	Mu KQ, Ren XJ, Yang H, et al. CRISPR-Cas12a-based diagnostics of wheat fungal diseases [J]. J Agric Food Chem, 2022, 70(23): 7240-7247.
[14]	Fang YX, Liu LJ, Zhao WY, et al. Rapid and sensitive detection of Verticillium dahliae from soil using LAMP-CRISPR/Cas12a technology [J]. Int J Mol Sci, 2024, 25(10): 5185.
[15]	Guo YF, Tan JJ, Jiao BB, et al. Establishment of a rapid detection system for Phytophthora syringae based on RPA/CRISPR-Cas12a [J]. Crop Prot, 2025, 190: 107106.
[16]	Nguyen LT, Rananaware SR, Pizzano BLM, et al. Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection [J]. Commun Med, 2022, 2: 7.
[17]	Dong JJ, Wang SJ, Xu WX, et al. Understanding the stability landscape of LbCas12a by deep analysis of stabilizing mutations and mutation combinations [J]. Protein Sci, 2025, 34(9): e70280.
[18]	Tong XH, Li TL, Zhang K, et al. Structure-guided design of Cas12a variants improves detection of nucleic acids [J]. Cell Insight, 2025, 4(2): 100228.
[19]	Hwang I, Song YH, Lee S. Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products [J]. Biosens Bioelectron, 2025, 267: 116859.
[20]	Nguyen LT, Smith BM, Jain PK. Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection [J]. Nat Commun, 2020, 11: 4906.
[21]	Li YY, University CM, Hu QF, et al. CrRNA conformation-engineered CRISPR-Cas12a system for robust and ultrasensitive nucleic acid detection [J]. Anal Chem, 2025, 97(6): 3617-3624.
[22]	Cheng ZH, Luo XY, Yu SS, et al. Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection [J]. Nat Commun, 2025, 16: 1166.
[23]	Son H, Park J, Hwang I, et al. Mg²⁺-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage [J]. Proc Natl Acad Sci U S A, 2021, 118(49): e2113747118.
[24]	Wang D, Ma DJ, Han JX, et al. CRISPR RNA array-guided multisite cleavage for gene disruption by Cas9 and Cpf1 [J]. ChemBioChem, 2018, 19(20): 2195-2205.
[25]	Su F, Wang S, Gao W, et al. Visual detection of multiple Fusarium species via RAA-CRISPR/Cas12a dual fluorescence-lateral flow assay [J]. J Future Foods, 2025
[26]	Chen JL, Huang YE, Xiao B, et al. Development of a RPA-CRISPR-Cas12a assay for rapid, simple, and sensitive detection of Mycoplasma hominis [J]. Front Microbiol, 2022, 13: 842415.
[27]	Pan JF, Deng F, Liu Z, et al. Construction of molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly and CRISPR-Cas12a [J]. Talanta, 2023, 255: 124210.
[28]	Nguyen GT, Schelling MA, Raju A, et al. CRISPR-Cas12a exhibits metal-dependent specificity switching [J]. Nucleic Acids Res, 2024, 52(16): 9343-9359.
[29]	Zhu FX, Yu H, Zhao Q. CRISPR/Cas12a-amplified aptamer switch microplate assay for small molecules [J]. Anal Chem, 2024, 96(17): 6853-6859.
[30]	Zhang YD, He BS, Zhang YR, et al. An aptasensor based on CRISPR/cas12a-mediated and Mn²⁺-assisted DNA motor cascade signalling amplification strategy for the detection of T-2 toxin [J]. Sens Actuat B Chem, 2025, 426: 137049.
[31]	Liu Y, Liu XY, Wei DY, et al. CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels [J]. Nat Commun, 2024, 15: 5014.