Direct Detection of dCas9-mediated Target Gene by Ion Concentration Polarization

Abstract

Our group has reported that nanofluidic preconcentration devices utilizing ion concentration polarization (ICP)1, 2 enables simultaneous separation and preconcentration of biomolecules3. Further development of the device which is the main topic of this work enlightens us that the behaviors of accumulated analyte can be categorized into “propagating” and “stacking” behavior depending on the critical mobility which is the function of intrinsic and extrinsic properties of materials. Thus, altering the mobility of target DNA using dCas9 realized optical and direct sequence-specific detection. Therefore, the proposed dCas9-mediated genetic detection methodology based on ICP would provide rapid and direct micro/nanofluidic platform for liquid biopsies. I. Introduction The-state-of-the-art bio- and nanotechnology have opened up an avenue to non-invasive liquid biopsy for identifying diseases from biomolecules in bloodstream, especially DNA. In this work, we combined sequence-specific-labeling scheme using mutated Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 without endonuclease activity (CRISPR/dCas9) and ion concentration polarization (ICP) phenomenon as a mechanism to selectively preconcentrate targeted DNA molecules for rapid and direct detection. II. Results Theoretical analysis on ICP phenomenon figured out a critical mobility, elucidating two distinguishable concentrating behaviors in the vicinity of a nanojunction; a stacking and a propagating behavior (Figure 1(a), 1(b)). Through the modulation of the critical mobility to shift those behaviors, the C-C chemokine receptor type 5 (CCR5) sequences were optically detected without PCR amplification (Figure 1(c), 1(d), 1(e)). III. Conclusion The proposed dCas9-mediated genetic detection methodology based on ICP would provide rapid and accurate micro/nanofluidic platform of liquid biopsies for disease diagnostics, especially PCR-free gene detection. Acknowledgements This work was supported by Korean Health Technology RND Project (HI13C1468 and HI14C0559). References S. J. Kim, Y.-A. Song and J. Han, Chem. Soc. Rev., 2010, 39, 912-922.S. J. Kim, Y.-C. Wang, J. H. Lee, H. Jang and J. Han, Phys. Rev. Lett., 2007, 99, 044501.J. Choi, K. Huh, D. J. Moon, H. Lee, S. Y. Son, K. Kim, H. C. Kim, J.-H. Chae, G. Y. Sung, H.-Y. Kim, J. W. Hong and S. J. Kim, RSC Advances, 2015, 5, 66178-66184. Fig. 1. Time-lapse images of each concentrating behavior of (a) Sulforhodamine B molecule (stacking regime, convection dominant case) and (b) Alexa 532 molecule (propagating regime, electromigration dominant case). (c) Target genetic sequence binding process of dCas9:sgRNA complex. Schematic for detecting target sequence by dCas9-mediated ICP platform (d) with target gene (positive) and (e) without target gene (negative). Figure 1