electroporation-mediated-delivery-cas9-ribonucleoproteins-mrna-into

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Clemson University

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne neglected tropical disease endemic to rural sub-Saharan Africa. Current methods of early detection in the affected rural communities generally begin with general screening using the card agglutination test for trypanosomiasis (CATT), a serological test. However, the gold standard for confirmation of trypanosomiasis remains the direct observation of the causative parasite, . Here, we present the use of dielectrophoresis (DEP) to enrich  parasites in specific locations to facilitate their identification in a future diagnostic assay. DEP refers to physical movement that can be selectively induced on the parasites when exposing them to electric field gradients of specific magnitude, phase and frequency. The long-term goal of our work is to use DEP to selectively trap and enrich  in specific locations while eluting all other cells in a sample. This would allow for a diagnostic test that enables the user to characterize the presence of parasites in specific locations determined  instead of relying on scanning a sample. In the work presented here, we report the characterization of the conditions that lead to high enrichment, 780% in 50 s, of the parasite in specific locations using an array of titanium microelectrodes.

Highly Localized Enrichment of  Parasites Using Dielectrophoresis.

Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.

Callie Stuart

Department of Bioengineering

Clemson University

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

Callie Stuart

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bioengineering

Clemson University

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

Department of Bioengineering