TY - JOUR T1 - Investigation of Cas9 antibodies in the human eye. JF - Nat Commun Y1 - 2022 A1 - Toral, Marcus A A1 - Charlesworth, Carsten T A1 - Ng, Benjamin A1 - Chemudupati, Teja A1 - Homma, Shota A1 - Nakauchi, Hiromitsu A1 - Bassuk, Alexander G A1 - Porteus, Matthew H A1 - Mahajan, Vinit B KW - Animals KW - Antibodies KW - CRISPR-Associated Protein 9 KW - CRISPR-Cas Systems KW - Humans KW - Mice KW - Streptococcus pyogenes KW - T-Lymphocytes AB -

Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.

VL - 13 IS - 1 ER - TY - JOUR T1 - CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa. JF - Mol Ther Y1 - 2016 A1 - Wu, Wen-Hsuan A1 - Tsai, Yi-Ting A1 - Justus, Sally A1 - Lee, Ting-Ting A1 - Zhang, Lijuan A1 - Lin, Chyuan-Sheng A1 - Bassuk, Alexander G A1 - Mahajan, Vinit B A1 - Tsang, Stephen H KW - Animals KW - Clustered Regularly Interspaced Short Palindromic Repeats KW - CRISPR-Cas Systems KW - Cyclic Nucleotide Phosphodiesterases, Type 6 KW - Disease Models, Animal KW - DNA Repair KW - Electroretinography KW - Exons KW - Gene Editing KW - Genetic Loci KW - Homologous Recombination KW - Mice KW - Mice, Transgenic KW - Mutation KW - Photoreceptor Cells, Vertebrate KW - Retinal Degeneration KW - Retinitis Pigmentosa KW - RNA, Guide AB -

Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.

VL - 24 IS - 8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/27203441?dopt=Abstract ER -