Single-molecule detection of CRISPR-Cas second messengers using nanopores
Second messengers are molecules with a central role in cellular signaling and communication. For the first time, an interdisciplinary team led by Sonja Schmid achieved the detection of CRISPR-Cas-produced messengers with single-molecule resolution using label-free nanopore detection, as published in the journal ACS Nano.
CRISPR-Cas systems are widely known for their gene editing capacity used in diverse biotech and biomedical applications. However, originally, CRISPR-Cas evolved in bacteria as an anti-viral defense system. Interestingly, the type III CIRSPR/Cas variants exert a unique second layer of defense: a cell with high viral load can commit suicide in an altruistic attempt to stop viral spread and thereby save its neighboring cells. Given the fatal outcome, this mechanism is tightly regulated by so-called secondary messenger molecules – specifically, cyclic oligo-adenylates (cOA) – but their precise role and, linked to that, their (stoichiometric) composition are still under study.
Counting molecules one-by-one
Previously, only large numbers (ensembles) of these cOA messengers were detectable which even then relied on expensive and bulky equipment (e.g. LC-MS). With the newly developed nanopore - neural network pipeline, spearheaded by PhD student David Fuentenebro Navas, it is now possible to detect, count, and identify even single cOA messenger molecules. The protein nanopores used in this work are nanoscale sensors for small molecules which are electronically detected as they pass through the nanopore. In contrast to the earlier expensive approach, the label-free nanopore procedure is fast, affordable, and potentially portable. Moreover, thanks to the single-molecule resolution of the approach, it allows researchers to identify even rare but often decisive molecules in mixtures, which are missed by conventional bulk assays.
The new nanopore pipeline allowed the authors to identify the hitherto unknown composition of CRISPR messenger molecules occurring in the bacterium Thermus thermophilus, with interesting biological implications. Beyond that, the new single-molecule detector could become useful for biomedical point-of-care devices, in the future.
Original Publication
David Fuentenebro Navas, Jurre A. Steens, Carlos de Lannoy, Ben Noordijk, Michael Pfeffer, Dick de Ridder, Raymond H.J. Staals, Sonja Schmid
Nanopores Reveal the Stoichiometry of Single Oligoadenylates Produced by Type III CRISPR-Cas
ACS Nano 202418 (26), 16505-16515 DOI: 10.1021/acsnano.3c11769
Further Information
Website research group Schmid