PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.

Generation and characterization of a Stargardt's disease-specific induced pluripotent stem cell line (LVPEIi008-A) with a homozygous nonsense mutation in exon 44 of ABCA4.

The Stargardt's Disease, Type 1 (STGD1) is associated with the loss of function mutations in ABCA4. This gene codes for a retina-specific, ATP-binding cassette (ABC) family transporter, involved in the transport of the key visual cycle intermediate, all-trans-retinaldehyde (atRAL), across the photoreceptor cell membranes. Here, we report the establishment of a patient-specific, iPSC line (LVPEIi008-A), that carries a homozygous nonsense mutation at (c.

Generation of two induced pluripotent stem cell lines (LVPEIi004-A and LVPEIi005-A) from probands with Leber Congenital Amaurosis 2 (LCA2) and harboring mutations in RPE65.

Leber Congenital Amaurosis 2 is an early onset retinal dystrophy that occurs due to mutation in RPE65 gene. Here, we report the generation of two patient specific induced pluripotent stem cell lines harboring nonsense mutations in exon 7 (c.646A > T) and exon 9 (c.

Generation of two induced pluripotent stem cell lines (LVPEIi007-B, LVPEIi008-B) from patients harboring homozygous mutation in ABCA4 (c.6088C>T) using non-integrative Sendai virus-based approach.

Mutations in ABCA4 gene leads to the most common form of an inherited retinal disease namely, the Stargardt disease, type 1. Here, we report the generation of two different patient-specific induced pluripotent stem cell lines (LVPEIi007-B and LVPEIi008-B), carrying an identical homozygous mutation, (c.6088C>T) within the exon 44 of ABCA4 gene.

Generation and characterization of three CRISPR/Cas9 edited RB1 null hiPSC lines for retinoblastoma disease modelling.

Complete loss of RB1 causes retinoblastoma. Here, we report the generation of three RB1 iPSC lines using CRISPR/Cas9 based editing at exon 18 of RB1 in a healthy control hiPSC line. The edited cells were clonally expanded, genotyped and characterized to establish the mutant lines.

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M).