CRISPR/Cas12a-Assisted Visual Logic-Gate Detection of Pathogenic Microorganisms Based on Water-Soluble DNA-Binding AIEgens

Abstract

Here, we developed a rapid, visual and double-checked Logic Gate detection platform for detection of pathogenic microorganisms by aggregation-induced emission luminogens (AIEgens) in combination with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas). DNA light-up AIEgens (1,1,2,2-tetrakis[4-(2-bromo-ethoxy) phenyl]ethene, TTAPE) was non-emissive but the emission was turned on in the presence of large amount of DNA produced by recombinase polymerase amplification (RPA). When CRISPR/Cas12a was added, all long-stranded DNA were cut leading to the emission quenched. Thus, a method that can directly observe the emission changes with the naked eye has been successfully constructed. The detection is speedy within only 20 min, and has strong specificity to the target. The result can be judged by Logic Gate. Only when the output signal is (1,0), does it represent the presence of pathogenic microorganisms in the test object. Finally, the method was applied to the detect pathogenic microorganisms in environmental water samples, which proved that this method has high selectivity, specificity and applicability for the detection of pathogenic microorganisms in environmental water samples.

Keywords: CRISPR-Cas12a; aggregation-induced emission (AIE); logic gate; pathogenic microorganisms; visual detection.

FIGURE 1

(A) Schematic diagram of TTAPE-CRISPR/Cas12a platform for detecting pathogenic microorganisms; (B) Logical gate schematic diagram and real table value; (C) The process of CRISPR/Cas12a to recognize specific DNA fragments of L. pneumophila; (D) Chemical structure of TTAPE.

FIGURE 2

The visual discrimination results under UV irradiation (365 nm) and PL spectra (λ_ex. = 350 nm) of (A) L. pneumophila positive sample; (B) E.coli positive sample; (C) S.aureus positive sample; (D) L. pneumophila negative sample; (E) E.coli negative sample; (F) S.aureus negative sample (a: +TTAPE, b: +TTAPE +Cas12a-RNP).

FIGURE 3

(A) The visual discrimination results under UV irradiation and gray scale images of L. pneumophila at different concentrations (a: +TTAPE, b: +TTAPE +Cas12a-RNP); (B) Gray value of L. pneumophila at different concentrations (the gray value is obtained by analyzing the gray image by image J). (C) Linear relationship of the fluorescence (I/I₀) and logarithm concentration of L. pneumophila.

FIGURE 4

(A) Diagram of the TTAPE-CRISPR/Cas12a platform for detecting environmental water samples; Gel electrophoresis diagram (B) and the visual discrimination results under UV irradiation (C) of L. pneumophila in environmental water samples (The target DNA length is 291 bp. “+” represents samples containing L. pneumophila, “−” represents samples without L. pneumophila. 1–3: air-conditioned water samples, 4–6: lake water samples, 7–9: tap water samples; a: +TTAPE, b: +TTAPE +Cas12a-RNP).