A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity

Figures

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  FIGURE 1. The primer‐based Cas12a detection system (PCDetection) for β‐thalassemia mutation detection. (A) Schematic diagram representing PCDetection. Primer 1 contains protospacer adjacent motif (PAM) and CRISPR RNA (crRNA) sequences (named Barcode), and the base located in the 3′‐end is modified with locked nucleic acid, which can increase the annealing temperature. When the template and primer binding sequences completely match, an amplicon with the Barcode is generated and detected by Cas12a, then yields fluorescence signals by cleaving ssDNA‐fluorophore‐quencher (FQ) probes. If there is a mismatch between the template and the3′‐end of primer binding sequence, there will be no amplification product and no fluorescence signal. (B) Schematic diagram representing detection of one variant of β‐thalassemia dominant mutation, HBB‐28, CD41‐42, and IVS‐II‐654, using PCDetection in a single test. The circles represent the components of the system. (C) Nine β‐thalassemia carriers were detected using specific PCDetection for the three β‐thalassemia mutations, HBB‐28, CD41‐42, and IVS‐II‐654. Each assay was performed in triplicates. (D) Schematic diagram representing detection of multiple variants…

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  FIGURE 2. Quantitative detection capability of Cas12a. (A) The fluorescence values of the sequentially diluted genomic DNA (gDNA) samples. (B) The linear relationship between gDNA sample concentration and fluorescence value. Δ, represents subtracted the background fluorescence. Error bars represent the means ± SD; n = 3.

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  FIGURE 3. The barcode‐based Cas12a detection system (BCDetection) used for detecting β‐thalassemia mutations. (A) Flow chart representing BCDetection workflow. Probe 1 contains a barcode sequence, including the PAM and crRNA sequences, Primer F sequence, and specific hybridized sequence. Probe 2 contains the Primer R binding sequence and specific hybridized sequence. When the template and probe binding sequence completely match, a ligated product with the targeted sequence of crRNA is generated by Primer F/R and detected by Cas12a, then yields fluorescence signals by cleaving ssDNA‐FQ probes. When there is a mismatch between the template and the 3′‐end of Probe 1 binding sequence, no ligated product is generated, and no fluorescence signal. (B) Cas12a fluorescence assay was used to detect the products generated under different reaction conditions for ligation and PCR amplification. L, ligation cycles; P, PCR cycles. Δ, represents subtracted the background fluorescence. (C) Schematic diagram representing detection of one variant of β‐thalassemia mutation using BCDetection in a single test. (D) Three β‐thalassemia carriers were detected using specific BCDetection for the three β‐thalassemia…

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  FIGURE 4. Quantitative detection capability of barcode‐based Cas12a detection system. (A) The fluorescence values of the sequentially diluted genomic DNA (gDNA) samples. (B) The linear relationship between the concentration of gDNA samples and fluorescence value in the range of 4–256‐fold dilutions. (B) The linear relationship between the gDNA concentration and fluorescence value in the range of 8–256‐fold dilutions. (D) The linear relationship between the concentration of gDNA samples and fluorescence value in the 16–256‐fold dilution range. Error bars represent the means ± SD; n = 3. Δ, represents subtracted the background fluorescence.

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  FIGURE 5. The two‐tube barcode‐based Cas12a detection (BCDetection) method used for survival motor neuron 1 gene (SMN1) copy number detection. (A) Schematic diagram representing SMN copy number detection. Theoretically, for normal individuals carrying two copies of SMN1 and albumin (ALB), after hybridization, ligation, and amplification, the copy number of amplicons of the two genes is approximately the same; furthermore, the final ratio of increment in fluorescence value of SMN1 to that of ALB is close to 1. In spinal muscular atrophy (SMA) carriers, the ratio is 0.5. In SMA patients, the ratio is near 0. (B) The change curves of fluorescence value obtained from normal individuals, SMA carriers, and SMA patients using BCDetection. Δ, represents subtracted the background fluorescence. Error bars indicate mean ± SEM; n = 3. (C) Comparison of the ratio of increment in SMN1 fluorescence value to ALB fluorescence value in normal individuals, SMA carriers, and SMA patients detected using BCDetection. Error bars indicate means ± SD; n = 20. ****, p < 0.0001.

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  FIGURE 6. The one‐tube barcode‐based Cas12a detection (BCDetection) method used for survival motor neuron 1 gene (SMN1) copy number detection. (A) Schematic diagram representing SMN copy number detection using the one‐tube BCDetection method. After hybridization, ligation, and PCR amplification in one tube, the amplicons were detected using albumin (ALB) and SMN1 probes targeted against crRNAs. Theoretically, for normal individuals carrying two copies of SMN1 and ALB, the final ratio of increment in fluorescence value of SMN1 to that of ALB is close to 1. In SMA carriers, the ratio is 0.5. In spinal muscular atrophy (SMA) patients, the ratio is 0. (B) The change curves of fluorescence value obtained from normal individuals, SMA carriers, and SMA patients using the one‐tube BCDetection method. Δ, represents subtracted the background fluorescence. Error bars indicate mean ± SEM; n = 3. (C) Comparison of the ratio of increment in SMN1 fluorescence value to ALB fluorescence value in normal individuals, SMA carriers, and SMA patients detected using BCDetection. Error bars indicate means ± SD; n = 20. ****, p < 0.0001.