Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection

Abstract

The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.

Fig. 1. Inhibition of cis- and trans-cleavage activities of LbCas12a by heparin sodium.

a Schematic diagram of heparin sodium regulating Cas12a cleavage activity. b Cis-cleavage activity characterized using a plasmid containing the f3l and b6r genes. This plasmid also includes a BsaI restriction site. c Fluorescence detection of LbCas12a cis-cleavage activity in the presence of 2 µg/mL heparin sodium, with crRNA targeting f3l or b6r. d, e Fluorescent characterization of cis-cleavage at various concentrations of heparin sodium. The aggregated f3l and b6r fluorescent probe templates are cleaved to generate fluorescence. f Fluorescence detection of LbCas12a trans-cleavage activity in the presence of 2 µg/mL heparin sodium, with crRNA targeting f3l or b6r. g, h Fluorescent characterization of trans-cleavage at various concentrations of heparin sodium, with FAM-CCCCCCCC-BHQ1 being cleaved to generate fluorescence. Data are shown as mean ± s.d. for n  =  3 biologically independent samples.

Fig. 2. Inhibition mechanism of Cas12a cleavage activity by heparin sodium.

a Schematic representation of the three potential stages of Cas12a activity inhibited by heparin sodium. b, c Impact of heparin sodium on Cas12a cleavage activity at three distinct stages when crRNA targets f3l (b) or b6r (c). d The binding affinity of crRNA for Cas12a in the presence of heparin sodium using EMSA. e ΔG of crRNA, heparin sodium, and S-free heparin with LbCas12a. f Root Mean Square Deviation (RMSD) of LbCas12a in different simulation systems. g–k, Electrostatic potential analysis between Cas12a and crRNA (g); Cas12a and heparin (h); Cas12a, crRNA, and heparin (i); Cas12a and S-free heparin (j); Cas12a, crRNA, and S-free heparin (k). Data are shown as mean ± s.d. for n  =  3 biologically independent samples.

Fig. 3. Heparin sodium-based one-pot Cas12a detection method.

a, b Schematic of the one-pot detection method, where RPA amplification and Cas12a-mediated fluorescence detection occur simultaneously within a single tube. b–d Characterization of the LoD of the RPA-Cas12a one-pot detection method without (b) and with 40 µg/mL heparin sodium (c) and the qPCR (d), targeting the f3l gene at concentrations of 0, 0.1, 1, 10, 100, 1000, 10000 aM. e–g LoD characterization of the RPA-Cas12a one-pot method targeting b6r without (e) and with the 40 µg/mL heparin sodium (f) and the qPCR (g). h, i At a target concentration of 10 fM, with Cas12a-crRNA (100 nM) targeting f3l (h) or b6r (i), the content of RPA amplification products at various times were analyzed in agarose gels. Samples without heparin sodium or Cas12a during the RPA amplification process were used as the controls. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. The schematics shown in Fig. 3a were created by figdraw.com.

Fig. 4. Characterizations of one-pot detection using classical and suboptimal PAMs with LbCas12a, AsCas12a, or AapCas12b.

a Validation of the SURVEY method when targeting f3l at 1 fM (1000 aM) with classical PAMs (TTTC and TTTG). bValidation of the SURVEY method when targeting b6r at 1 fM with classical PAMs (TTTC and TTTG). c–e Validation of the SURVEY method when targeting f3l or b6r with suboptimal PAMs (CTTT (c), ATTA (d) and TTAA (e)). f Schematic illustration of heparin sodium enhancing one-pot detection with various Cas12 proteins. g Characterization of the SURVEY method for targeting f3l or b6r when LbCas12a was substituted with AsCas12a. h Characterization of the SURVEY method for targeting f3l or b6r when LbCas12a was substituted with AapCas12b. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. Statistical significance was analyzed using a two-tailed t test: ns, p > 0.05. The schematics shown in Fig. 4f were created by figdraw.com.

Fig. 5. Specificity and sensitivity of the SURVEY method in mpox pseudovirus detection.

a, b Specific characterization of the SURVEY method using environmental pathogens such as SARS-CoV-2, adenovirus, influenza A virus, Mycoplasma pneumoniae, Pseudomonas aeruginosa, and Helicobacter pylori as targets (crRNA targeting f3l of mpox (a), and crRNA targeting b6r of mpox (b)).c, d Detection of various concentrations of pseudovirus (carrying f3l (c) or b6r (d) genes) introduced into saliva using both qPCR and the SURVEY method. The threshold was set as the negative control plus three times its standard deviation. Fluorescence values exceeding this threshold were defined as positive, while those below were defined as negative. e, f Sensitivity (also known as the true positive rate) defined as: Sensitivity = True Positives/(True Positives + False Negatives). Specificity (also known as the true negative rate) defined as: Specificity = True Negatives/(True Negatives + False Positives). qPCR serves as the gold standard for determining negative and positive results. Data are shown as mean ± s.d. for n  =  3 biologically independent samples.

Fig. 6. Detection of influenza A virus with the SURVEY method.

a–c LoD analysis for the synthetic M2 protein coding gene (DNA) using the RPA-Cas12a one-pot detection method without (a) and with (b) 40 µg/mL heparin sodium, and qPCR (c) at target concentrations of 0, 0.1, 1, 10, 100, 1000, 10000 aM. d–f LoD analysis for transcribed mRNA targets using the RT-RPA-Cas12a one-pot detection method without (d) and with (e) 10 µg/mL heparin sodium, and RT-qPCR (f) at target concentrations of 0, 0.1, 1, 10, 100, 1000, 10000 aM. g Schematic representation of detection of human saliva and sputum samples. h Analysis of samples from all 33 patients using RT-qPCR and the SURVEY method. RT-qPCR was performed for 40 cycles. For SURVEY detection, the threshold was set to the negative control fluorescence value plus three times its standard deviation; samples exceeding this threshold were classified as positive, while those below were considered negative. i Specificity and sensitivity analysis for samples detection using the SURVEY method, with RT-qPCR serving as the gold standard for determining negative and positive results. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. Statistical significance was analyzed using a two-tailed t test: ns, p > 0.05. The schematics shown in Fig. 6g were created by figdraw.com.

Fig. 7. Detection of the SARS-CoV-2 with the SURVEY method.

a–c LoD analysis for the synthetic N protein coding gene (DNA) using the RPA-Cas12a one-pot detection method without (a) and with (b) 40 µg/mL heparin sodium, and qPCR (c) at target concentrations of 0, 0.1, 1, 10, 100, 1000, 10000 aM. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. d–f LoD analysis for transcribed mRNA targets using the RT-RPA-Cas12a one-pot detection method without (d) and with (e) 10 µg/mL heparin sodium, and RT-qPCR (f) at target concentrations of 0, 0.1, 1, 10, 100, 1000, 10000 aM. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. g Schematic representation of SARS-CoV-2 detection in wastewater samples from 10 provinces in China. h Analysis of samples from all 25 influents and 25 effluents using RT-qPCR and the SURVEY method. RT-qPCR was performed for 40 cycles. For SURVEY detection, the threshold was set to the negative control fluorescence value plus three times its standard deviation; samples exceeding this threshold were classified as positive, while those below were considered negative. Statistical significance was analyzed using a two-tailed t test: ns, p > 0.05. The schematics shown in (g) were created by figdraw.com.