A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.

CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs.

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

No Sex Differences in the Frequencies of Common Single Nucleotide Polymorphisms Associated with Age-Related Macular Degeneration.

PURPOSE: Since some studies have reported differences in the association of age-related macular degeneration (AMD) with biological sex, we set out to determine whether the difference in the disease susceptibility is afforded by common single nucleotide polymorphisms (SNPs) associated with AMD. METHODS: We genotyped 2067 Caucasian subjects from the Age-Related Eye Disease Study cohort for commonly associated AMD SNPs, including those in CFH (rs1061170, rs1410996, and rs3766404), ARMS2 (rs10490924), and C3 (rs2230199) using either a Sequenom MassARRAY MALDI-TOF mass spectrometer or using Taqman genotyping reagents. A Cox proportional hazards model was used to determine the effect of genotype, age, sex, and smoking status on the development of AMD. RESULTS: All tested SNPs genotyped are associated strongly with AMD (p < 0.0001), in concordance with previous studies. However, we found no observable differences in any of the SNPs studied when categorized by sex. Interactions between SNPs and sex were found to be not statistically significant (p = 0.38-0.79). CONCLUSIONS: The difference between male and female incidence of AMD is not explained by the most commonly AMD-associated SNPs, though it does not exclude the possibility that other, less common SNPs contribute to this difference.

Functional single nucleotide polymorphism in IL-17A 3' untranslated region is targeted by miR-4480 in vitro and may be associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. Genetic factors contributing to AMD include single nucleotide polymorphisms (SNPs) in immune-related genes including CFH, C2, CFI, C9, and C3, thus implicating these pathways in AMD pathogenesis. MicroRNAs (miRNAs) are powerful regulators of gene expression and execute this function by binding to the 3' untranslated region (3'UTR) of target mRNAs, leading to mRNA degradation. In this study, we searched for the possible association of SNPs in the 3'UTR region of IL-17A, a gene implicated in AMD pathogenesis without any previous SNP association with AMD. Using two independent sample cohorts of Caucasian subjects, six SNPs in the IL-17A 3'-UTR were selected for genotyping based on bioinformatic predictions of the SNP effect on microRNA binding. The SNP rs7747909 was found to be associated with AMD (P < 0.05) in the NEI cohort, using a dominant model logistic regression. Luciferase reporter gene assays and RNA electrophoretic mobility shift assays were performed using ARPE-19 cells to confirm the preferential binding of microRNAs to the major allele of the SNP. Our findings support the hypothesis that microRNA-mediated gene dysregulation may play a role in the pathogenesis of AMD.

Responses of Multipotent Retinal Stem Cells to IL-1ß, IL-18, or IL-17.

Purpose. To investigate how multipotent retinal stem cells (RSCs) isolated from mice respond to the proinflammatory signaling molecules, IL-1ß, IL-18, and IL-17A. Materials and Methods. RSCs were cultured in a specific culture medium and were treated with these cytokines. Cell viability was detected by MTT assay; ultrastructure was evaluated by transmission electron microscopy; expression of IL-17rc and proapoptotic proteins was detected by immunocytochemistry and expression of Il-6 and Il-17a was detected by quantitative RT-PCR. As a comparison, primary mouse retinal pigment epithelium (RPE) cells were also treated with IL-1ß, IL-18, or IL-17A and analyzed for the expression of Il-6 and Il-17rc. Results. Treatment with IL-1ß, IL-18, or IL-17A decreased RSC viability in a dose-dependent fashion and led to damage in cellular ultrastructure including pyroptotic and/or necroptotic cells. IL-1ß and IL-18 could induce proapoptotic protein expression. All treatments induced significantly higher expression of Il-6 and Il-17rc in both cells. However, neither IL-1ß nor IL-18 could induce Il-17a expression in RSCs. Conclusions. IL-1ß, IL-18, and IL-17A induce retinal cell death via pyroptosis/necroptosis and apoptosis. They also provoke proinflammatory responses in RSCs. Though IL-1ß and IL-18 could not induce Il-17a expression in RSCs, they both increase Il-17rc expression, which may mediate the effect of Il-17a.

Animal models of age-related macular degeneration and their translatability into the clinic.

Age-related macular degeneration (AMD) is a leading cause of blindness in people over the age of 55. Despite its common nature, the etiology of the disease involves both genetic and environmental factors, the interaction of which is not fully understood. Animal models, including the mouse, rat, rabbit, pig and non-human primate, have been developed to study various aspects of the disease and to evaluate novel therapies; however, no single model has been developed to emulate all aspects of the disease. This review will discuss the various existing models of AMD, their strengths and limitations and examples of their use in current AMD research.