Self‐Assembled DNA Nanoclews for the Efficient Delivery of CRISPR–Cas9 for Genome Editing

Abstract

CRISPR-Cas9 represents a promising technology for genome editing, yet means of safe and efficient delivery remain to be fully realized. Here, we report a novel delivery vehicle to deliver the Cas9 protein and single-guide RNA simultaneously based on DNA nanoclews, yarn-like DNA nanoparticles synthesized by rolling circle amplification. The bio-inspired vehicles efficiently loaded Cas9/single-guide RNA complexes and delivered the complexes to the nuclei of human cells, allowing targeted gene disruptions while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile platform that could be adapted for delivering other DNA-binding proteins or for functional nucleic acids.

dCas9: A Versatile Tool for Epigenome Editing

“…Alternatively, cells stably expressing dCas9-effector proteins can be complemented by delivery of sgRNA, but stable expression is difficult to achieve when using primary cells or multicellular organisms. Another option is dCas9 delivery using in vitro assembled sgRNA-(d)Cas9 ribonucleoproteins (RNPs), an approach which was successful for dCas9-VP64 and Cas9 genome editing applications (Cho et al, 2013b;Kim et al, 2014;Ramakrishna et al, 2014;Sun et al, 2015;Zuris et al, 2015;D' Astolfo et al, 2015). Finally, a potential hurdle for implementation in living organisms is the fact that the (d) Cas9 protein elicits an immunogenic responses due to its exogenous origin (Wang et al, 2015a).…”

Intracellular Delivery of Proteins By Nanocarriers

“…These are yarn-like DNA nanoparticles that are synthesized by rolling circle amplification with palindromic sequences for self-assembly of NPs. Recently, Gu and colleagues [38] have used NCs to deliver CRISPR-Cas9 into mammalian cells. Specific DNA NCs were designed complimentary to the CRISPRsgRNA it will carry.…”

A genome editing primer for the hematologist

“…134 Other forms of nonviral in vivo delivery, such as ultrasound with microbubbles, cationic lipid complexes, and DNA nanoparticles, have been investigated. [135][136][137] Given the pace of advances, even in vivo delivery of genome editing reagents targeted directly to hematopoietic cells including HSCs seems conceivable. In contrast to their typical use via ex vivo transduction, lentiviral and nonlentiviral vectors may directly transduce HSCs following in vivo intraosseous or intravenous delivery.…”