RT Journal Article
SR Electronic
T1 CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.11.08.467750
DO 10.1101/2021.11.08.467750
A1 Enrique Lin-Shiao
A1 Wolfgang G. Pfeifer
A1 Brian R. Shy
A1 Mohammad Saffari Doost
A1 Evelyn Chen
A1 Vivasvan S. Vykunta
A1 Jennifer R. Hamilton
A1 Elizabeth C. Stahl
A1 Diana M. Lopez
A1 Cindy R. Sandoval Espinoza
A1 Alexander E. Dejanov
A1 Rachel J. Lew
A1 Michael G. Poirer
A1 Alexander Marson
A1 Carlos E. Castro
A1 Jennifer A. Doudna
YR 2021
UL http://biorxiv.org/content/early/2021/11/09/2021.11.08.467750.abstract
AB 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 Sentence CRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cellsCompeting Interest StatementThe authors have filed a patent application covering the intellectual property included in this work. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Vertex, Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences, The Column Group and Inari. J.A.D. is a Director at Johnson & Johnson and Tempus and has research projects sponsored by Biogen, Pfizer, AppleTree Partners, and Roche. A.M. is a compensated co-founder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. was a compensated member of the scientific advisory board at PACT Pharma and was a compensated advisor to Juno Therapeutics and Trizell. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, and PACT Pharma. A.M. has received fees from Merck and Vertex and is an investor in and informal advisor to Offline Ventures. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. B.R.S, V.S.V., and A.M. hold patents pertaining to but not resulting from this work.