Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01%

Chen, Kena; Dai, Ling; Jie, Zhao; Deng, Mei; Song, Lin; Bai, Dan; Wu, You; Zhou, Xi; Yang, Yujun; Yang, Shuangshuang; Zhao, Lin; Chen, Xueping; Li, Junjie

doi:10.1016/j.talanta.2023.124674

Cited by 1 publication

(2 citation statements)

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“…Other approaches to remove the PAM limitation include the use of AaCas12b, which sets short PAM (5′-TTN-3′) 55 and the assay reaction with T m -reducing reagent at 48 °C. 56 Finally, we evaluated the sensitivity of the Cas12-crRNA/ ssDNA reporter-immobilized spot. Figure 6 shows the cleavage ratios of the pVenus-N1-targeting spot in the presence of various concentrations of pVenus-N1 after 2 h of incubation and washing.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Since the PAM sequence in target dsDNA is necessary to be recognized by Cas12, it can be easily inserted into any dsDNA sequences by using a PAM-fused primer during PCR, as shown in the experimental procedures and Table S1. Other approaches to remove the PAM limitation include the use of AaCas12b, which sets short PAM (5′-TTN-3′) and the assay reaction with T m -reducing reagent at 48 °C …”

Section: Resultsmentioning

confidence: 99%

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Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex Double-Stranded DNA Detection

Shigemori,

Fujita,

Tamiya

et al. 2023

Bioconjugate Chem.

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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12 (Cas12) system is attracting interest for its potential as a next-generation nucleic acid detection tool. The system can recognize double-stranded DNA (dsDNA) based on Cas12-CRISPR RNA (crRNA) and induce signal transduction by collateral cleavage. This property is expected to simplify comprehensive genotyping. Here, we report a solid-phase collateral cleavage (SPCC) reaction by CRISPR/Cas12 and its application toward one-pot multiplex dsDNA detection with minimal operational steps. In the sensor, Cas12-crRNA and single-stranded DNA (ssDNA) are immobilized on the sensing surface and act as enzyme and reporter substrates, respectively. We also report a dual-target dsDNA sensor prepared by immobilizing Cas12-crRNA and a fluorophore-labeled ssDNA reporter on separate spots. When a spot captures a target dsDNA sequence, it cleaves the ssDNA reporter on the same spot and reduces its fluorescence by 42.1–57.3%. Crucially, spots targeting different sequences do not show a reduction in fluorescence, thus confirming the one-pot multiplex dsDNA detection by SPCC. Furthermore, the sequence specificity has a two-base resolution, and the detectable concentration for the target dsDNA is at least 10–9 M. In the future, the SPCC-based sensor array could achieve one-pot comprehensive genotyping by using an array spotter as a reagent-immobilizing method.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%