The
use of CRISPR/Cas9 systems in genome editing has
been limited
by the inability to efficiently deliver the key editing components
to and across tissues and cell membranes, respectively. Spherical
nucleic acids (SNAs) are nanostructures that provide privileged access
to both but have yet to be explored as a means of facilitating gene
editing. Herein, a new class of CRISPR SNAs are designed and evaluated
in the context of genome editing. Specifically, Cas9 ProSNAs comprised
of Cas9 cores densely modified with DNA on their exteriors and preloaded
with single-guide RNA were synthesized and evaluated for their genome
editing capabilities in the context of multiple cell lines. The radial
orientation of the DNA on the Cas9 protein surface enhances cellular
uptake, without the need for electroporation or transfection agents.
In addition, the Cas9 proteins defining the cores of the ProSNAs were
fused with GALA peptides on their N-termini and nuclear localization
signals on their C-termini to facilitate endosomal escape and maximize
nuclear localization and editing efficiency, respectively. These constructs
were stable against protease digestion under conditions that fully
degrade the Cas9 protein, when not transformed into an SNA, and used
to achieve genome editing efficiency between 32 and 47%. Taken together,
these novel constructs and advances point toward a way of significantly
broadening the scope of use and impact of CRISPR-Cas9 genome editing
systems.
Proteins are exquisite nanoscale building blocks: molecularly pure, chemically addressable, and inherently selective for their evolved function. The organization of proteins into single crystals with high positional, orientational, and translational order results in materials where the location of every atom can be known. However, controlling the organization of proteins is challenging due to the myriad interactions that define protein interfaces within native single crystals. Recently, we discovered that introducing a single DNA−DNA interaction between protein surfaces leads to changes in the packing of proteins within single crystals and the protein−protein interactions (PPIs) that arise. However, modifying specific PPIs to effect deliberate changes to protein packing is an unmet challenge. In this work, we hypothesized that disrupting and replacing a highly conserved PPI with a DNA−DNA interaction would enable protein packing to be modulated by exploiting the programmability of the introduced oligonucleotides. Using concanavalin A (ConA) as a model protein, we circumvent potentially deleterious mutagenesis and exploit the selective binding of ConA toward mannose to noncovalently attach DNA to the protein surface. We show that DNA association eliminates the major PPI responsible for crystallization of native ConA, thereby allowing subtle changes to DNA design (length, complementarity, and attachment position) to program distinct changes to ConA packing, including the realization of three novel crystal structures and the deliberate expansion of ConA packing along a single crystallographic axis. These findings significantly enhance our understanding of how DNA can supersede native PPIs to program protein packing within ordered materials.
A novel helicate complex [Fe II 2 L 3 ] 4+ resulted from subcomponent self-assembly of a C 3-symmetric triamine, 2-formylpyridine and octahedral iron(II) in CH 3 CN, which was confirmed by ESI-MS measurement. After the addition of selected planar aromatic molecules in CD 3 CN solution, shifts of 1 H NMR signals of the helicate were investigated. The results revealed that the size, functional group and symmetry of guest molecules remarkably influence the interaction patterns.
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