Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection

Abstract

CRISPR technology holds significant promise for advancing nucleic acid assays. However, current CRISPR diagnostic techniques, reliant on indiscriminate trans-cleavage mechanisms, face challenges in developing multiplex detection formats. Moreover, chaotic trans-cleavage activity often results from mismatched targets, leading to specificity issues. To address these limitations, here we exploit a double-key recognition mechanism based on CRISPR-Cas12a cis-cleavage and invasive hybridization identification of released sticky-end DNA products. By integrating multiplexed nucleic acid amplification, the double-key Cas12a detection mechanism, and a lateral flow detection platform, we develop a method termed Cas12a cis-cleavage mediated lateral flow assay (cc-LFA). We demonstrate that the cc-LFA exhibited superior specificity compared to three mainstream trans-cleavage-based CRISPR diagnostic techniques, achieving single-base resolution detection free from high-concentration wild-type DNA background interference. cc-LFA is also applied for highly specific detection of multiple respiratory pathogen samples and precise multiplexed detection of nine high-risk human papillomavirus (HPV) subtypes, achieving over 90% sensitivity and 100% specificity, respectively. Additionally, we present a portable device to automate nucleic acid amplification and strip detection procedures, showcasing the potential of cc-LFA for future applications in decentralized laboratory scenarios.

Fig. 1. Exploiting Multiplexed and highly specific nucleic acid assay based on Cas12a cis-cleavage.

a Scheme of conventional Cas12a trans-cleavage nucleic acid assay. Conventional Cas12a nucleic acid assays based on the trans-cleavage mechanism encounter a challenge in implementing multiplexed detection formats, whether using fluorescence or strip detection methods. Conventional Cas12a nucleic acid assay also presents specificity issues due to the trans-cleavage activity does not strictly depend on perfect crRNA-targeted DNA pairing. PAM-distal DNA bases typically exhibit low specificity to trans-cleavage. b Cas12a cis-cleavage based nucleic acid assay. In this assay, the target DNA is first checked by Cas12a cis-cleavage, and then the released PAM-distal DNA product with sticky ends is further identified via invasion hybridization. Such a detection principle consists of two checkpoints, making it highly specific. In addition, invasion hybridization extends target recognition beyond the crRNA recognition region, further enhancing specificity. By designing multiple crRNAs and invasion probes, it is possible to achieve highly specific and multiplexed nucleic acid detection based on this double-key recognition mechanism.

Fig. 2. Feasibility and specificity of Cas12a cis-cleavage mediated invasion hybridization.

a LbCas12a cis-cleavage mediates the generation of sticky-end DNA products. Using the ASFV DNA sequence as a target, sequencing results indicated that the cis-cleavage product contains a 6-base sticky end. b 5′-overhang dsDNA is not a preferred substrate for LbCas12a trans-cleavage. Fluorescence experiments indicate that the cleavage efficiency of ssDNA substrates is much higher than that of 5′-overhang dsDNA. The data collection time of the bar chart was 20 min. c Validation of invasion hybridization using fluorescently labeled 5′-overhang dsDNA substrate and ssDNA invasion probes. The 5′-overhang dsDNA contains a 5-bp sticky end. Fluorescence experiments showed that invasion hybridization triggers displacement of quenched-group-labeled DNA, resulting in significant fluorescence enhancement. d, e Evaluation of sequence dependence of invasion hybridization by fluorescence experiments. Invasion DNA probes contain single-base or two-base mutations that cross all locations. The fluorescence intensity was compared to a perfectly matched invasion probe hybridization. Data are presented as mean values +/− SD (n = 3, independent replicates) in (b, d and e), and the error bars represent SD. a.u. represents arbitrary units. Source data are provided as a Source Data file.

Fig. 3. Development of cc-LFA platform.

a cc-LFA is a simple nucleic acid detection technology without the need for optical detection instruments. Nucleic acid targets are amplified by biotinylated primers, followed by Cas12a cis-cleavage and AuNP-DNA probe invasion hybridization, and finally captured by the strip for color development. b Reaction components deletion experiments for corroborating the cc-LFA concept. The right panel shows the color development results and the quantification of the test and control line intensity. c Scheme of three kinds of cc-LFA detection modes. In operation mode 1, the gold nanoparticle probes were pre-embedding on the conjugate pad. After 10 min of reaction at 37 °C, the CRISPR system containing the amplified target product were added to the conjugate pad for a 10 min strand invasion reaction at room temperature before proceeding with the lateral flow assay. In operation mode 2, the gold nanoparticle probes were added to the CRISPR system after cis-cleavage, followed by a 10-minute incubation at room temperature before proceeding with the lateral flow assay. Operation mode 3, the amplified target product, CRISPR reaction system, and gold nanoparticle probes into a single tube, which was incubated for 10 min at 37 °C before directly proceeding to the lateral flow assay. d Test results obtained under the three cc-LFA test modes. e Test results shown from all-in-one operation mode 3 with different reaction times. f The analytical sensitivity of cc-LFA was evaluated by detecting different concentrations of African swine fever virus DNA. The dotted line referred to the detection limit, which was determined by the intensity of the NTC plus three times the standard deviation. Data are presented as mean values +/− SD (n = 3, independent replicates) in (d, e and f), and the error bars represent SD. For f, statistical analysis was conducted using a two-tailed ttest. Statistical significance is indicated as follows: n.s. = no significance with P > 0.05, and the asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). The accurate P-value was listed above the corresponding error bar. NTC represents non-template control. The test line strength is extracted using Image J software. a.u. represents arbitrary units. Source data are provided as a Source Data file.

Fig. 4. Evaluation and comparison of analytical specificity of cc-LFA with other mainstream CRISPR nucleic acid assays.

a and b show the LbCas12a crRNA and its paired target. The targets tested include perfectly matched sequences and sequences with 2 consecutive base mutations or single-base mutations at different locations. c cc-LFA platform is adapted for the detection of two-base mismatched sequence (d) and single-base mismatched sequence (e). f Cas12a trans-cleavage based lateral flow assay is adapted for detection of two-base mismatched sequence (g) and single-base mismatched sequence (h). i Cas12a trans-cleavage based fluorescence assay is adapted for the detection of two-base mismatched sequence (j) and single-base mismatched sequence (k). l Cas9 based CASLFA platform is adapted for the detection of two-base mismatched sequence (m) and single-base mismatched sequence (n). The complete paired, single-base, or two-base mutated targets used in the above four methods were the same bridge PCR products. Data are presented as mean values +/− SD (n = 3, independent replicates) and the error bars represent SD. The test line strength is extracted using Image J software. PM represents a perfectly matched sequence. a.u. represents arbitrary units. Source data are provided as a Source Data file.

Fig. 5. Multiplexed cc-LFA platform for detecting clinical respiratory pathogen samples.

a Development of dual-gene detection of SARS-CoV-2 RNA based on cc-LFA. b Three kinds of typical test situations based on dual-gene cc-LFA. Quantification of the test line intensity is also shown in the below panel. c Evaluation of the analytical sensitivity and specificity for detecting SARS-CoV-2 RNA based on dual-gene cc-LFA. Data are presented as mean values +/− SD (n = 3, independent replicates) and the error bars represent SD. NTC represents non-template control. For c, statistical analysis was conducted using a two-tailed t test. Statistical significance is indicated as follows: n.s. = no significance with P > 0.05, and the asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.). The accurate P-value was listed above the corresponding error bar, with the O gene above and the N gene below. a.u. represents arbitrary units. d Dual-gene cc-LFA was employed to detect 45 clinical SARS-CoV-2 samples. These clinical samples were confirmed using CFDA-approved RT-qPCR kits, and their Ct values were listed correspondingly. e Sensitivity performance is stated based on the detection of samples with Ct values below 35 and 38, respectively. f Development of cc-LFA for simultaneously detecting three respiratory pathogens. g Typical 8 kinds of test situations based on three-gene cc-LFA. Quantification of the test line intensity is also shown in the below panel. h Three-gene cc-LFA was employed to detect 112 clinical respiratory disease samples. These clinical samples were confirmed using CFDA-approved RT-qPCR kits, and their Ct values were listed correspondingly. Sensitivity performance of detecting IBV (i), IAV (j), and SARS-CoV-2 (k) are stated based on the detection of samples with Ct values below 35 and 38, respectively. Source data are provided as a Source Data file.

Fig. 6. Development of nine-gene cc-LFA platform.

a Orthogonal experimental analysis to evaluate the specificity of crRNAs and invasion probes designed for the identification of 9 high-risk HPV subtypes. The targets used are all biotinylated PCR products. b Orthogonal experimental analysis to evaluate the specificity of Cas12a trans-cleavage based fluorescence assay for identification of 9 high-risk HPV subtypes. c Scheme of the nine-gene cc-LFA platform for assaying high-risk HPV subtypes. d Single HPV subtype and four combinations of multiple HPV subtypes were detected by nine-gene cc-LFA. #4 represents HPV 16, 18, 31, and 33. #5 represents HPV 16, 11, 18, 31, and 33. #7 represents HPV 16, 11, 6, 18, 31, 33, and 58. #9 represents HPV 16, 11, 6, 18, 31, 33, 45, 52 and 58. Specifically, the HPV-16 detected by T1 Line is a biotinylated PCR amplification product, while the remaining eight T-lines detect the other eight HPV subtypes, each represented by PCR products containing barcode sequences with a C3 spacer modification. e 32 potential HPV infected clinical samples were detected by nine-gene cc-LFA. These samples are confirmed by a clinically adopted PCR reagent. f Sensitivity and specificity analysis. NTC represents non-template control. Source data are provided as a Source Data file.

Fig. 7. Development of a portable cc-LFA device.

a The external rendering of a portable device, measuring 86 mm × 149 mm × 135 mm in length × width × height. b The internal structure diagram of the portable device and the structure of the thin-film sample pad. c The internal workflow of the device. d The operation process of using portable devices for nucleic acid testing. e The detection limit of the POC device was tested with 200000, 20000, 2000, 200, 20, 2 copies of HPV16 DNA as template. f Quantification of test results with different copy numbers. The dotted line indicates the detection limit, which is determined by the NTC strength plus three times the standard deviation. Data are presented as mean values +/− SD (n = 3, independent replicates) and the error bars represent SD. For f, statistical analysis was conducted using a two-tailed t test. Statistical significance is indicated as follows: n.s. = no significance with P > 0.05, and the asterisks (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). The accurate P value was listed above the corresponding error bar. NTC represents a non-template control. Image J software was used to extract the test line strength. a.u. represents arbitrary units. g 50 clinical samples were used to evaluate the detection performance of the portable device, including 2 co-infected samples with HPV16 and HPV18. The yellow square indicates the HPV16 positive sample identified by the portable device, the blue square indicates the HPV18 positive sample identified by the portable device, the gray square indicates the positive sample identified by qPCR, and the white square indicates negative samples. The sample 26 with Ct 34.04 was not detected. h Receiver operating characteristic (ROC) analysis was performed on the results of 50 clinical samples detected by a POC device, and the AUC value was 0.9872. Source data are provided as a Source Data file.