{
  "abstract": "CRISPR-Cas12a is a programmable RNA-guided endonuclease whose activation by double-stranded DNA normally depends on the recognition of an adjacent protospacer-adjacent motif (PAM), which constrains target selection in diagnostic applications. Here, we divide the DNA activator into PAM-proximal (Pp) and PAM-distal (Pd) duplexes, spatially separating the PAM requirement from the interrogation of a PAM-less target sequence. FRET, electrophoretic mobility shift, fluorescence polarization, and functional assays indicate that either fragment can associate with Cas12a, but that robust productive activation requires the PAM-containing Pp duplex together with the Pd duplex. Our data further support a model in which PAM-mediated protein-DNA contacts stabilize an assembly-competent complex and facilitate binding of the PAM-less Pd target capture. Weakening hybridization between the guide and Pp with a chimeric DNA/RNA increased the dependence on PAM-mediated stabilization and improved structural selectivity in the Pp region and single-nucleotide specificity in the Pd region. The resulting split-duplex architecture provides an ultraspecific platform for resolving single-nucleotide polymorphisms in the PAM-less DNA strand.",
  "abstract_shown": true,
  "affiliations": [
    "Department of Molecular Medicine, Laval University, Quebec, QuebecG1V 0A6, Canada.",
    "Centre de Recherche du CHU de Québec, Quebec, QuebecG1V 4G2, Canada.",
    "Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ONM5S 3M2, Canada.",
    "Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ONM5S 1A1, Canada."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 12,
  "authors": [
    {
      "affs": [
        0,
        1
      ],
      "name": "Gabriel Lamothe",
      "orcid": "0000-0003-4468-7775"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Felix Veillette"
    },
    {
      "affs": [
        2,
        3
      ],
      "name": "Idorenyin A Iwe",
      "orcid": "0000-0003-3438-7517"
    },
    {
      "affs": [
        2
      ],
      "name": "Ariel Corsano"
    },
    {
      "affs": [
        2
      ],
      "name": "Suman Shrestha"
    },
    {
      "affs": [
        2
      ],
      "name": "Serena Singh"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Camille Bouchard"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Kelly Godbout"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Yaoyao Lu"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Joel Rousseau"
    },
    {
      "affs": [
        2,
        3
      ],
      "name": "Keith Pardee",
      "orcid": "0000-0002-3812-8013"
    },
    {
      "affs": [
        0,
        1
      ],
      "name": "Jacques P Tremblay"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/4cghxe89x8tpm.bib",
  "cited_by": 0,
  "doi": "10.1021/acs.analchem.6c03855",
  "fulltext_url": "https://europepmc.org/articles/PMC13670894",
  "id": "4cghxe89x8tpm",
  "issns": [
    "0003-2700",
    "1520-6882"
  ],
  "landing_url": "https://europepmc.org/article/MED/42916138",
  "language": "eng",
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|CRISPR and Genetic Engineering"
    ]
  ],
  "openalex": "W7214770935",
  "pdf_url": "https://europepmc.org/articles/PMC13670894?pdf=render",
  "pmcid": "PMC13670894",
  "pmid": "42916138",
  "published": "2026-10-01",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/4cghxe89x8tpm.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:20:36Z",
      "native_id": "MED:42916138",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42916138"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|CRISPR and Genetic Engineering",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex.",
  "title_text": "Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:20:36Z",
  "url": "https://research.draconitic.com/paper/4cghxe89x8tpm",
  "venue": "Analytical chemistry",
  "versions": []
}