The Construction Of CRISPR System Delivered By Inorganic Nanocarriers And Its Application In Transient Gene Editing

The CRISPR system gene editing technology provides a promising approach for treating disease.However,delivery of CRISPR system mainly relies on adeno-associated viruses(AAVs).The inherent defects of AAVs,such as immunogenicity,low loading capacity and integration risk caused by long-term expression of Cas9 protein sg RNA,which limits their further clinical application.Therefore,the development of novel nano vectors mediated CRISPR system is important for transient gene editing.In this paper,inorganic nanomaterials with high loading capacity and stability were used as carriers to deliver CRISPR/Cas9 plasmid and SpCas9 variant without PAMs restriction(SpRY)plasmid into mammalian cells to achieve transient gene editing.1.Graphene Oxide Nanoparticles Combined with CRISPR/Cas9 System Enable Efficient Inhibition of Pseudorabies VirusCRISPR/Cas9 transient gene editing mediated by inorganic nanocarriers can effectively avoid the risk of integration caused by viral vectors.This study described an application where GONs enable combined inhibition of PRV through delivery of a CRISPR/Cas9 system for targeted cleaving of a PRV genome and direct interaction with viral particles.Cell study showed that sheeted GONs can carry negatively charged CRISPR/Cas9 p DNA to form spherical GONs-CRISPR complexes around 50 nm,which enables efficient intracellular endocytosis and transient expression of Cas9 protein under serum conditions.Viral study showed that GONs alone had antiviral activity and combined with CRISPR/Cas9 p DNA(GONS-CRISPR)could significantly reduce PRV progeny in host cells by nearly 4,000-fold.This GONs-based strategy may expand the use of CRISPR systems in antiviral applications.2.Gene editing of Duchenne muscular dystrophy using biomineralization-based SpCas9 variant nanoparticlesThe wild type CRISPR/Cas9 has the limitation of NGG PAM,which hinders gene editing in many pathogenic gene loci.In this study,an inorganic biomineralized PAMLess Cas9 variant(SpRY)nanoparticles(Bm-SpRY NPs)was developed for DMD gene editing in vitro and in vivo.This work describes a method for the synthesis of simple biomineralized nanoparticles with high SpRY p DNA encapsulation efficiency.In vitro results showed that Bm-SpRY NPs had obvious advantages of well biocompatibility and protecting from enzymatic degradation and efficient delivery under high serum conditions.Cell study demonstrated that Bm-SpRY NPs enable cell uptake,lysosomes escape and nucleus transport.Meanwhiles,Bm-SpRY NPs mediated gene editing is a transient process without risk of integration.In this work,Bm-SpRY NPs were used to edit four different PAMs in the splice receptor region of exon 51 of DMD gene and found that target region with TAG PAM had the highest editing efficiency(20.6%)and significant preference mutation(base A addition).In vivo study demonstrated that intramuscular injection of Bm-SpRY NPs in mice enable DMD gene mutations without tissue damage.This study can expand the application prospect of CRISPR system in DMD treatment.In summary,according to the many defects of CRISPR system delivered by AAVs,this article designs(1)graphene oxide nanocarrier loading CRISPR/Cas9 plasmid to inhibit PRV virus replication in PK15 cells.In order to lift limitation of NGG PAM existing in CRISPR/Cas9,(2)The biomineralized nanomaterials were designed to deliver the SpCas9variant(SpRY)plasmid without PAMs restriction,which realized the gene editing of four different PAMs in exon 51 of DMD gene in cells,and further gene editing in mouse muscle tissue.These inorganic nanocarriers can efficiently carry CRISPR systems and mediate a transient and safe gene editing,which is expected to expand the application of inorganic nanomaterials in the field of disease.