Targeted gene knockout by direct delivery of zinc-finger nuclease proteins

Abstract

Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

Figure 1. ZFN proteins are cell permeable and induce targeted mutagenesis in human cells

(a) Diagram of ZFN protein and electrostatic potential of the ZFN protein surface colored from dark red (− 5 kT/e) to white (0 kT/e) to dark blue (+ 5 kT/e). (b) Schematic representation of the fluorescence reporter system used to evaluate cellular penetration of ZFN proteins. (c–e) Percentage of EGFP-positive cells as determined by flow cytometry in reporter cells treated with increasing amounts of ZFN proteins (c), subjected to consecutive treatments with 0.2 μM ZFN proteins (d), or subjected to three consecutive treatments with increasing amounts of ZFN proteins (e), in all cases using either standard or transient hypothermic conditions. Error bars indicate s.d. (n = 3). (f) Representative sequence analysis of the EGFP locus from isolated EGFP-positive cells. Multiple deletions (dashes) and insertions (lowercase) induced by NHEJ repair are aligned to the cleavage site (wt).

Figure 2. Modification of endogenous human genes by direct delivery of ZFN proteins

(a) Frequency of endogenous CCR5 gene disruption in HEK293, THP1, HDF and CD4⁺ T cells subjected to three consecutive treatments with ZFN proteins using transient hypothermic conditions, as determined by the Surveyor nuclease assay. Black arrow indicates specific cleavage product. (b) DHFR production measured by fluorescein-methotrexate binding in CHO cells subjected to three consecutive treatments with ZFN proteins using standard or transient hypothermic conditions. Mock treated CHO cells exhibited approximately 1% reduction in functional DHFR (not shown). (c) Proliferation of HEK293, HDF and THP1 cells treated with ZFN proteins targeting the CCR5 gene and containing Sharkey mutations. (d) Proliferation of CHO cells treated with ZFN proteins targeting the DHFR gene and utilizing a variety of specialized cleavage domains. Values normalized to mock treated cells. Error bars indicate s.d. (n = 3).