High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells.

ABSTRACT: Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells. Two plasmids encoding enhanced green and red fluorescent proteins (EGFP and ERFP), respectively, were successfully electroporated into attached HeLa cells on a 169-microwell array chip with transfection efficiencies of 71.6?±?11.4% and 62.9?±?2.7%, and a cell viability above 95%. We also successfully conducted selective electroporation of sgRNA into 293T cells expressing the Cas9 nuclease in a high-throughput manner and observed the four-fold increase of the GFP intensities due to the repair of the protein coding sequences mediated by the CRISPR/Cas9 system. This study proved that this HiCEP system has the great potential to be used for arrayed functional screens with genome-wide CRISPR libraries on hard-to-transfect cells in the future.

SUBMITTER: Bian S 

PROVIDER: S-EPMC5304186 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature