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

Lin-Shiao E, Pfeifer WG, Shy BR, Saffari Doost M, Chen E, Vykunta VS, Hamilton JR, Stahl EC, Lopez DM, Sandoval Espinoza CR, Deyanov AE, Lew RJ, Poirer MG, Marson A, Castro CE, Doudna JA
Source: Nucleic Acids Res
Publication Date: (2022)
Issue: 50(3): 1256-1268
Research Area:
Basic Research
Cells used in publication:
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: kidney
Culture Media:
4D-Nucleofector® 96-well Systems

RNP electroporation: Cas9 RNPs were formulated as previously described (29). In brief, immediately prior to electroporation sgRNA (IDT) was resuspended in IDT duplex buffer to 80 µM and combined with polyglutamic acid sodium salt (Alamanda Polymers, CAS #26247-79-0) dissolved in ddH2O to 125 mg/ml at a ratio of 1:0.8 sgRNA to PGA. sgRNA/PGA mix and Cas9-NLS (UC Berkeley QB3 MacroLab, stock at 40 µM) were then mixed at a 2:1 molar ratio and incubated at 37°C for 15 min to form Cas9 RNPs at a final concentration of 13.3 µM. Four microliters of complexed RNPs were then incubated with different amounts of HDR templates for 5 min prior to electroporation. Electroporation was performed using a 96-well format 4D nucleofector (Lonza) with 200 000 cells per well. HEK293T cells were electroporated with the SF buffer and the CM-130 pulse code, K562 cells with SF buffer and the FF-120 pulse code, and T cells with the P3 buffer and the EH-115 pulse code. Cells were immediately resuspended in pre-warmed media, incubated for 20 min and transferred to culture plates.


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.