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Peer-reviewedCC BY-NC-NDAnalytical chemistry

Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex.

  • Gabriel Lamothe1,2
  • Felix Veillette1,2
  • Idorenyin A Iwe3,4
  • Ariel Corsano3
  • Suman Shrestha3
  • Serena Singh3
  • Camille Bouchard1,2
  • Kelly Godbout1,2
  • Yaoyao Lu1,2
  • Joel Rousseau1,2
  • Keith Pardee3,4
  • Jacques P Tremblay1,2
4 affiliations
  1. 1Department of Molecular Medicine, Laval University, Quebec, QuebecG1V 0A6, Canada.
  2. 2Centre de Recherche du CHU de Québec, Quebec, QuebecG1V 4G2, Canada.
  3. 3Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ONM5S 3M2, Canada.
  4. 4Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ONM5S 1A1, Canada.
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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.

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Cite

BibTeXRIS
BibTeX
@article{lamothe2026split,
  title = {{Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex.}},
  author = {Gabriel Lamothe and Felix Veillette and Idorenyin A Iwe and Ariel Corsano and Suman Shrestha and Serena Singh and Camille Bouchard and Kelly Godbout and Yaoyao Lu and Joel Rousseau and Keith Pardee and Jacques P Tremblay},
  journal = {Analytical chemistry},
  year = {2026},
  month = oct,
  doi = {10.1021/acs.analchem.6c03855},
  url = {https://europepmc.org/article/MED/42916138},
  note = {CC BY-NC-ND},
}
RIS
TY  - JOUR
TI  - Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex.
AU  - Gabriel Lamothe
AU  - Felix Veillette
AU  - Idorenyin A Iwe
AU  - Ariel Corsano
AU  - Suman Shrestha
AU  - Serena Singh
AU  - Camille Bouchard
AU  - Kelly Godbout
AU  - Yaoyao Lu
AU  - Joel Rousseau
AU  - Keith Pardee
AU  - Jacques P Tremblay
PY  - 2026
DA  - 2026/10/01
JO  - Analytical chemistry
DO  - 10.1021/acs.analchem.6c03855
UR  - https://europepmc.org/article/MED/42916138
AB  - 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.
SN  - 0003-2700
SN  - 1520-6882
ER  -