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Role of GUCA1C in Primary Congenital Glaucoma and in the Retina: Functional Evaluation in Zebrafish.
Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.
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Assessment of Miniature AsCas12f1 Variants for Gene Editing and Activation.
Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V-F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking. This study tested combinations of four AsCas12f1 protein variants and six sgRNA scaffolds for their gene editing and transcription activation efficiencies. The protein variant AsCas12f1-HKRA performed the best in gene editing and activation when paired with sgRNA-en_v2.1 scaffold. Furthermore, we validated a super miniature gene activator by fusing a small activation domain to AsCas12f1-HKRA. Our findings recommend using AsCas12f1-HKRA and sgRNA-en_v2.1 for gene editing and activation applications.
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Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipulation.
Advances in gene editing tools such as CRISPR/Cas9 have made precise in vivo gene editing possible, opening up avenues of research into somatic cell reprograming to study adult stem cells, homeostasis, and malignant transformation. Here we describe a method for CRISPR/Cas9 mediated in vivo gene editing, in combination with Cre-based lineage tracing via electroporation in the mouse oviduct. This method facilitates the delivery of multiple plasmids into oviduct epithelial cells, sufficient for studying homeostasis and generation of high-grade serous ovarian cancer (HGSOC) models.
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RNA gene editing in the eye and beyond: The neglected tool of the gene editing armatorium?
RNA editing allows correction of pathological point mutations without permanently altering genomic DNA. Theoretically targetable to any RNA type and site, its flexibility and reversibility makes it a potentially powerful gene editing tool. RNA editing offers a host of potential advantages in specific niches when compared to currently available alternative gene manipulation techniques. Unlike DNA editors, which are currently too large to be delivered in vivo using a viral vector, smaller RNA editors fit easily within the capabilities of an adeno-associated virus (AAV). Unlike gene augmentation, which is limited by gene size and viral packaging constraints, RNA editing may correct transcripts too long to fit within a viral vector. In this article we examine the development of RNA editing and discuss potential applications and pitfalls. We argue that, although in its infancy, an RNA editing approach can offer unique advantages for selected retinal diseases.
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Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner.
BACKGROUND: Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure. RESULTS: Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid. CONCLUSIONS: These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9.
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Therapeutic Gene Editing with CRISPR: A Laboratory Medicine Perspective.
Therapeutic gene editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system offers significant improvements in specificity and programmability compared with previous methods. CRISPR editing strategies can be used ex vivo and in vivo with many theoretic disease applications. Off-target effects of CRISPR-mediated gene editing are an important outcome to be aware of, minimize, and detect. The current methods of regulatory approval for personalized therapies are complex and may be proved inefficient as these therapies are implemented more widely. The role of pathologists and laboratory medicine practitioners is vital to the clinical implementation of therapeutic gene editing.
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CRISPR/Cas-Mediated Gene Activation as a Versatile Tool for Treatment of Inherited Retinal Dystrophies.
CRISPR/Cas-mediated genome editing is an effective and attractive tool for the treatment of diseases or genes that cannot be adequately covered by gene replacement strategies. The first FDA-approved AAV-vector- and CRISPR/Cas-based clinical trials were each designed for the treatment of a subtype of an inherited retinal dystrophy, underscoring the importance of ophthalmic diseases in the field of gene (editing) therapies. This and most other pre-clinical therapeutic CRISPR/Cas approaches are typically designed for the treatment of single mutations. For the treatment of larger patient cohorts, however, mutation- or ideally gene-independent approaches appear to be more suitable. CRISPR/Cas-mediated transcriptional activation of genes (CRISPRa) is a promising approach to achieve these ambitious goals. In this minireview, focusing on inherited retinal dystrophies, we will discuss recent developments, advantages and limitations, and future prospects of CRISPRa as a therapeutic tool.
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CRISPR: a journey of gene-editing based medicine.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is one of the hallmark of biological tools, contemplated as a valid and hopeful alternatives to genome editing. Advancements in CRISPR-based technologies have empowered scientists with an editing kit that allows them to employ their knowledge for deleting, replacing and lately "Gene Surgery", and provides unique control over genes in broad range of species, and presumably in humans. These fast-growing technologies have high strength and flexibility and are becoming an adaptable tool with implementations that are altering organism's genome and easily used for chromatin manipulation. In addition to the popularity of CRISPR in genome engineering and modern biology, this major tool authorizes breakthrough discoveries and methodological advancements in science. As scientists are developing new types of experiments, some of the applications are raising questions about what CRISPR can enable. The results of evidence-based research strongly suggest that CRISPR is becoming a practical tool for genome-engineering and to create genetically modified eukaryotes, which is needed to establish guidelines on new regulatory concerns for scientific communities.
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CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations.
Gene therapy carries the potential to treat more than 10,000 human monogenic diseases and benefit an even greater number of complex polygenic conditions. The repurposing of CRISPR/Cas9, an ancient bacterial immune defense system, into a gene-editing technology has armed researchers with a revolutionary tool for gene therapy. However, as the breadth of research and clinical applications of this technology continues to expand, outstanding technical challenges and ethical considerations will need to be addressed before clinical applications become commonplace. Here, we review CRISPR/Cas9 technology and discuss its benefits and limitations in research and the clinical context, as well as ethical considerations surrounding the use of CRISPR gene editing.
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Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms.
Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a "proof of concept" for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.
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