(A-15) Mechano-Cas12a Assisted Tension Sensor (MCATS) for Massively Amplified Cell Traction Force Measurements

Introduction:: The ability for cells to generate mechanical forces is central to a wide range of biological processes ranging from immunology to coagulation and plays an important role in numerous pathologies such as cancer. Therefore, developing methods to quantify cell-generated force has important biomedical applications. A central challenge in this field is that molecular forces that are sensed and transduced by cells are fairly weak, at the scale of piconewtons (pN) and are highly transient and infrequent. Here, we report the development of Mechano-Cas12a Assisted Tension Sensor (MCATS) that utilizes CRISPR-Cas12a to transduce and amplify the cumulative number of molecular force events generated by cells. We demonstrate the power of MCATS by detecting the forces generated by as few as ~10³ human platelets in a high-throughput manner. MCATS can be used to rapidly generate personalized dose-response curves and IC₅₀ for blood samples treated with FDA-approved drugs such as aspirin, eptifibatide(integrilin®), 7E3(Reopro®), and ticagrelor (Brelinta®). We also found that MCATS detected platelet dysfunction following cardiopulmonary bypass (CPB) which strongly correlated with the need for platelet transfusion.

Materials and Methods:: Specifically, we developed a Mechano-Cas12a Assisted Tension Sensor (MCATS), which is an ultrasensitive fluorescence-based assay to detect the aggregate number of molecular force events. In MCATS, the activator is a ssDNA anchored to a surface, such as a glass slide. The activator is concealed by hybridization to a complementary strand that is in turn conjugated to a peptide such as, cyclo-Arg-Gly-Asp-Phe-Lys (cRGDfK), or any protein ligand specific to the cell receptor of interest. When cells are seeded on this surface, surface receptors such as integrins bind to the cRGDfK ligand on the duplex and apply forces. Forces that exceed the mechanical tolerance of the duplex lead to its rupture, exposing the activator (bottom strand) and thus triggering Cas12a nuclease activity. Upon activation, Cas12a will indiscriminately and catalytically cleave fluorogenic single stranded DNA reporter for amplification. Because Cas12a is highly efficient, its activation generates a massive fluorescence signal output that can be measured using a conventional fluorometer or plate reader for facile and high throughput readout.

Results, Conclusions, and Discussions:: MCATS is a platform technology and can be applied to study many different types of cells and different biological processes. We used MCATS to investigate the forces generated by human platelets because of the importance of mechanical forces in platelet function. Indeed, recent studies using micropatterned polymer structures showed that platelets forces can be used to detect underlying genetic clotting disorders, and for predicting trauma-induced coagulopathy. Because MCATS only requires ~5 uL of blood or less to conduct each measurement, a typical blood draw (~5 mL) allows one to run ~1000 assays in a rapid manner. We leveraged this capability to screen the activity of a panel of clinically approved anti-platelet drugs such as aspirin, integrilin, abciximab, and Brelinta. These experiments demonstrate the potential of MCATS for personalized tailoring of anti-coagulant drugs and may help guide therapeutic intervention in the clinic. We also applied MCATS to detect platelet dysfunction following cardiopulmonary bypass (CPB). In a pilot study of seven CPB patients, we found that the change in MCATS signal strongly correlated with the need for platelet transfusion. More broadly, MCATS offers an accurate, rapid, and cost-efficient method to detect the integrated number of molecular force events generated by cells and will hence open the door to integrating mechanical measurements into the clinic.

Acknowledgements (Optional): :

References (Optional): :