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

Abstract

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000’s of bases are 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 (RNP) binding sites on the DNA nanostructures to increase shuttling of structures 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 (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large 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 measuring biophysical properties, into cell nuclei.Teaser SentenceCRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cells

Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

“…(2020) Ultralong ssDNA encoded with multiple functional sites Proton triggered release of Hhal enzyme causes cleavage of carrier DNA at encoded cleavage sites Co-delivery of sgRNA/Cas9 and DNAzyme inside lysosomes Li et al. (2022) Gene-based DNA origami Folding of gene-length ssDNA into DNA origami Co-delivery of entire genes and sgRNA/Cas9 Lin-Shiao et al. (2022) Other functions Tile-like DNA origami frame Binding sites in the hollow of the frame allow controlled suspension of target from two points Studying the Cas cleavage of relaxed and restrained dsDNA targets in real time with high-speed AFM Räz et al.…”

“…Another direction to integrate CRISPR/Cas systems and programmable DNA nanostructures has been recently signposted by the disruptive approach ( Lin-Shiao et al., 2022 ). Their proposal opens the door to DNA origami nanostructures that are designed to enhance or suppress DNA integration (depending on the application, see the previous sections), or even direct homologous recombination.…”

“…(D) adapted with permission from ( Abe et al., 2021 ); Copyright (2021) by Royal Society of Chemistry. (E) adapted with permission from ( Lin-Shiao et al., 2022 ); Published (2022) Oxford Academic Press. …”

“…In a very recent accomplishment, Lin-Shiao et al. (2022) designed a DNA nanostructure carrier approach for more efficient transport of even longer gene sequences into human cells.…”

“…Potential immunogenicity of Cas proteins must also be addressed in therapeutic applications ( Charlesworth et al., 2019 ). The field of DNA nanotechnology is well placed to address the Cas-gRNA (guide RNA) delivery challenge, and indeed has very recently been employed to do so ( Lin-Shiao et al., 2022 ).…”