CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

Abstract

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

Conformational Preferences at the Glycosidic Linkage of Saccharides in Solution as Deduced from NMR Experiments and MD Simulations: Comparison to Crystal Structures

“…A complex pattern of inter-residue hydrogen bonding was observed for the α-(1!4)-linked disaccharide being dependent on both the ψ torsion angle at the glycosidic linkage and the rotameric distributions of hydroxymethyl torsion angles. For the α-(1!3)-linked disaccharide containing a glucosyl residue at the reducing end the conformation from MD simulations and corresponding disaccharide elements identified in the PDB agreed closely to the crystal structure of the disaccharide as a tetrahydrate, whereas for α-(1!3)-linked disaccharide containing a galactosyl residue Glycosidic torsion angle conformations in crystal structures of compounds 1 [23] and 2 [24] are indicated by red crosses and a database search, using the glycan fragment database (GFDB), [44,96] at the reducing end the conformation from MD simulations deviated to the crystal structure of the disaccharide devoid of water molecules. The results obtained underscore the complementarity of crystal structures, MD simulations and NMR experiments in understanding of conformational aspects and flexibility at the glycosidic linkage of oligo-and polysaccharides that often are linked to proteins or lipids in the form of glycoconjugates, molecules essential in a wider scope of biology and living organisms.…”

“…Figure 13. Zoom-in of conformational space populated for the glycosidic torsion angles ϕ and ψ in 1-3 (a-c, respectively) color-coded as in figure 4.Glycosidic torsion angle conformations in crystal structures of compounds 1[23] and 2[24] are indicated by red crosses and a database search, using the glycan fragment database (GFDB),[44,96] of the Protein Data Bank for structures containing α-l-Fucp-(1!3)-d-Glcp (a) and α-d-Glcp-(1!4)-d-Galp (c) is indicated by black circles.…”

DNA Nanobarrel‐Based Drug Delivery for Paclitaxel and Doxorubicin

“…DNA nanostructure as a drug carrier can also overcome obstacles in regular drug delivery system such as stability, precise localization of target molecules, and the use of DNA nanostructure for drug delivery enables its translation from structural design to specific applications. Furthermore, recent reports showed the robustness of DNA origami for gene delivery, [38,39] reiterating the capacity of using DNA nanostructure as carrier materials for biomedical applications. Overall, the accurate positioning arising from DNA programmability spanning molecular to macroscope scale represents one indispensable advancement in nanotechnology and demands more systematic investigations for future nanomedicine design.…”

CRISPR/Cas systems combined with DNA nanostructures for biomedical applications

“…For example, Doudna's group used DNA origami to build a 18-helix DNA nanostructure that simultaneously encodes an integral human gene and contains CRISPR/Cas9 ribonucleoprotein binding sites for loading. This DNA nanostructure serves as a template for gene integration through CRISPR mediated homology-directed repair (HDR) 99 (Fig. 9(A)).…”