Supramolecular Nanosubstrate‐Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system has rapidly shifted from its natural role as an RNAguided genetic adaptive immune system in prokaryotes to a robust site-specific gene editing method. [1] The CRISPR/Cas9 system consists of two critical components, that is, the Cas9 endonuclease and a short, single-guide RNA (sgRNA), which form a Cas9•sgRNA ribonucleoprotein (RNP) complex. [2] Based on a simple basepairing mechanism, the RNP identifies and cuts a targeted DNA in the genome, leading to a double-strand break (DSB) at the specified location. [3] Endogenous DNA repair mechanisms, for example, the nonhomologous end joining (NHEJ) pathway, lead to insertions or deletions (indels) [4] that result in gene disruption. Gene disruption via CRISPR/Cas9 editing has been frequently applied for knocking down genes in cell lines and animal models. [5] Alternatively, complete gene knockout can be achieved via CRISPR/Cas9-mediated The clustered regularly interspaced short palindromic repeats (CRISPR)associated protein 9 (CRISPR/Cas9) is an efficient and precise geneediting technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Doseand time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.

CRISPR/Cas9 systems: Delivery technologies and biomedical applications

“…Ban et al. [78] devised a supramolecular substrate-mediated delivery (SNMD) approach by leveraging existing supramolecular nanoparticle (SMNP) carriers. They encapsulated RNP onto SMNP carriers, leading to the formation of RNP⊂SMNPs through self-assembly.…”

Synthetic nanoparticles for the delivery of CRISPR/Cas9 gene editing system: classification and biomedical applications

“…This provides a strategy to treat Duchenne muscular dystrophy. 85 Customized polymorphic nanoparticles can encapsulate large-sized pDNA. To engineer flexible dendritic copolymers, an incredibly controllable synthetic technique was devised using click chemistry based on G5 PAMAM.…”

“…4c). 85 Optimized SMNPs mediate the efficient delivery of Cas9 RNP target dystrophin gene in induce pluripotent stem cells (iPSC), MSCs as well as hAC16. This provides a strategy to treat Duchenne muscular dystrophy.…”

Delivering the CRISPR/Cas9 system for engineering gene therapies: Recent cargo and delivery approaches for clinical translation

“…In one study, SMNPs encapsulating plasmid encoding Cas9 and a guide targeting a safe-harbor locus and, separately, a plasmid encoding GFP plus a functional donor induced the correction of RS1 in vitro following intravitreal injection in a mouse model ( Chou et al, 2020 ). Nanowire-grafted SMNPs were optimized to package and deliver RNPs as well, inducing successful disruption of the dystrophin gene and knock-in of the HBB gene ( Yang et al, 2020 ; Ban et al, 2021 ). Similarly, Wan et al (2020) used disulfide-bridged biguanidyl adamantane (Ad-SS-GD) with CD-PEI as components for supramolecular assembly for the packaging and delivery of Cas9 RNPs and successfully induced nearly 16% editing in SW-280 cells.…”