Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody-and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

Pericyte loss is an early characteristic change in diabetic retinopathy (DR). Despite accumulating evidence that hyperglycemia-induced angiopoietin 2 (Ang2) has a central role in pericyte loss, the precise molecular mechanism has not been elucidated. This study investigated the role of Ang2 in pericyte loss in DR. We demonstrated that pericyte loss occurred with Ang2 increase in the diabetic mouse retina and that the source of Ang2 could be the endothelial cell. Ang2 induced pericyte apoptosis via the p53 pathway under high glucose, whereas Ang2 alone did not induce apoptosis. Integrin, not Tie-2 receptor, was involved for Ang2-induced pericyte apoptosis under high glucose as an Ang2 receptor. High glucose changed the integrin expression pattern, which increased integrin α3 and β1 in the pericyte. Furthermore, Ang2-induced pericyte apoptosis in vitro was effectively attenuated via p53 suppression by blocking integrin α3 and β1. Although intravitreal injection of Ang2 induced pericyte loss in C57BL/6J mice retina in vivo, intravitreal injection of anti-integrin α3 and β1 antibodies attenuated Ang2-induced pericyte loss. Taken together, Ang2 induced pericyte apoptosis under high glucose via α3β1 integrin. Glycemic control or blocking Ang2/integrin signaling could be a potential therapeutic target to prevent pericyte loss in early DR.