Christopher K. Fuller

Microfabricated Multi-Frequency Particle Impedance Characterization System

We have developed a microfabricated flow-through impedance characterization system capable of performing AC, multi-frequency measurements on cells and other particles. The sensor measures both the resistive and reactive impedance of passing particles, at rates of up to 100 particles per second. Its operational bandwidth approaches 10 MHz with a signal-to-noise ratio of approximately 40 dB. Particle impedance is measured at three or more frequencies simultaneously, enabling the derivation of multiple particle parameters. This constitutes an improvement to the well-established technique of DC particle sizing via the Coulter Principle. Human peripheral blood granulocyte radius, membrane capacitance, and cytoplasmic conductivity were measured (r = 4.1 μm, Cmem = 0.9 μF/cm2, σint = 0.66 S/m) and were found to be consistent with published values.

Mapping the genomic landscape of CRISPR-Cas9 cleavage

RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.