ArticleCommunications biology2024
Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform.
Article in Communications biology, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed.
- Combining Asymmetric PCR with PAM-Independent Cas12a Analysis of Single-Stranded Amplicons for On-Site Nucleic Acid Testing.Biosensors · 2026Article
- Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.Biochemical and biophysical research communications · 2026Article
- Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping.Nature communications · 2026Article
- CRISPR diagnostics: from trans-nuclease activity to cancer diagnosis.Cell & bioscience · 2026Review
- CrisprPr: a hybrid-driven framework for CRISPR/Cas9 off-target prediction with analysis of prior-information updates.Briefings in bioinformatics · 2026Article
- One-step detection of Chlamydia psittaci using recombinase polymerase amplification and CRISPR/Cas12b.AMB Express · 2026Article
- A One-Pot RPA-CRISPR/Cas12a Assay for Rapid Genus-Level Detection of Babesia spp. in Ticks and Livestock Blood Samples.Transboundary and emerging diseases · 2026Article
- CRISPR-based point-of-care diagnostics for viral hepatitis: from molecular design to clinical implementation.Frontiers in bioengineering and biotechnology · 2026Review
- A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System.Biosensors · 2025Article
- Advancements and Applications of Split Technology in CRISPR/Cas12a: Transforming Molecular Diagnostics and Biosensing.Biosensors · 2025Review
- PAM-Independent Cas12a Detection of Specific LAMP Products by Targeting Amplicon Loops.International journal of molecular sciences · 2025Article
- Research progress on the application of RPA-CRISPR/Cas12a in the rapid visual detection of pathogenic microorganisms.Frontiers in cellular and infection microbiology · 2025Review
Corrections and comments
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Authors and funding
13 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indicate that Cas12a specificity depends on the number, type, location, and distance of mismatches within the R-loop. We also find that introducing a wobble base pair at position 14 of the R-loop does not affect the free energy change when the spacer length is truncated to 17 bp. Therefore, we develop a new universal specificity enhancement strategy via iterative crRNA design, involving truncated spacers and a wobble base pair at position 14 of the R-loop, which tremendously increases specificity without sacrificing sensitivity. Additionally, we construct a PAM-free one-pot detection platform for SARS-CoV-2 variants, which effectively distinguishes SNV targets across various GC contents. In summary, our work reveals new insights into the specificity mechanism of Cas12a and demonstrates significant potential for in vitro diagnostics.
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