Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules

We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabili...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 116; no. 16; pp. 7899 - 7904
Main Authors Cao, Yuhong, Ma, Enbo, Cestellos-Blanco, Stefano, Zhang, Bei, Qiu, Ruoyi, Su, Yude, Doudna, Jennifer A., Yang, Peidong
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 16.04.2019
Subjects
BASIC BIOLOGICAL SCIENCES
Biological Sciences
Biomolecules
Cell membranes
Cell Survival
Cell viability
Cytological Techniques - methods
Electric contacts
Electric potential
Electric pulses
Electroporation
Electroporation - instrumentation
Electroporation - methods
Equipment Design
Fabrication
Gene Editing - methods
genome engineering
HeLa Cells
Humans
Intracellular
intracellular delivery
Intracellular Space - metabolism
Macromolecules
nanopore
Nanopores
nanotechnology
Nucleic acids
Physical Sciences
Porosity
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Ribonucleic acid
Ribonucleoproteins
RNA
Toxicity
Transfection - methods
Voltage
Water purification
nanotechnology
electroporation
intracellular delivery
nanopore
genome engineering
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Summary:We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.
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Keck Foundation
AC02-05CH11231; 89208-31150-44- X-IQJED
USDOE Office of Science (SC)
1Y.C. and E.M. contributed equally to this work.
Reviewers: H.P., Harvard University; and B.T., University of Chicago.
Contributed by Peidong Yang, February 25, 2019 (sent for review November 1, 2018; reviewed by Hongkun Park and Bozhi Tian)
Author contributions: Y.C., E.M., S.C.-B., and P.Y. designed research; Y.C., E.M., S.C.-B., R.Q., and Y.S. performed research; Y.C., E.M., B.Z., R.Q., and J.A.D. contributed new reagents/analytic tools; Y.C., E.M., R.Q., Y.S., and P.Y. analyzed data; J.A.D. and P.Y. supervised the research; and Y.C., E.M., S.C.-B., B.Z., Y.S., J.A.D., and P.Y. wrote the paper.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1818553116