Gene editing of Duchenne muscular dystrophy using biomineralization-based spCas9 variant nanoparticles

The CRISPR/Cas9 mediated genome editing have provided a promising strategy to correct multiple muta-tions of Duchenne muscular dystrophy (DMD). However, the delivery of CRISPR/Cas9 system into mam-malian cell for DMD gene editing mainly relies on adeno associated virus (AAV)-mediated transport. Meanwhile, the protospacer adjacent motif (PAM) requirement of wild-typed Cas9 protein causing the target sites for exon splice acceptor site are restricted to limited regions. Here, we developed a biomin-eralized PAMLess Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo . This method described a facile synthesis of biomineralized NPs with high SpRY pDNA encapsulation efficiency. In vitro results show that the Bm-SpRY NPs have the obvious advantages of well biocompati-bility and protecting SpRY pDNA from enzyme degradation and efficient delivery under high serum con-dition. Cell studies demonstrated that Bm-SpRY NPs enable rapid cellular uptake, endo-lysosomes escape and nucleus transport. Meanwhiles, the DMD gene editing via Bm-SpRY NPs pathway is transient process without genomic integration. We evaluated multiple target regions with different PAMs for the DMD exon 51 splice acceptor site through Bm-SpRY NPs method and found that the target region with TAG PAM has the highest editing efficiency and significant preferential mutation. In vivo results show that intramuscu-lar injection of Bm-SpRY NPs enable DMD gene mutation in muscle tissue without tissue damage. This study may extend the advanced application of CRISPR system for DMD therapy. Statement of significanceThe gene editing technology of CRISPR/Cas9 provides an effective treatment strategy for the Duchenne muscular dystrophy (DMD) therapy. However, the delivery of CRISPR system in mammalian cell mainly relies on viral mediated transport and the NGG or NAG requirement of wild-typed Cas9 protein limits the target region in DMD gene. Here, the present study provides a biomineralized PAM Less Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo . This study may extend the application of CRISPR system for DMD gene therapy.(c) 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Automated summary

The CRISPR/Cas9 gene editing technology provides a promising strategy for Duchenne muscular dystrophy (DMD) therapy. This study introduces biomineralized nanoparticles (Bm-SpRY NPs) as a method for efficient DMD gene editing in vitro and in vivo, with advantages of biocompatibility and high delivery efficiency.