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.

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease.

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini.

Coronary Disease Association With ADAMTS7 Is Due to Protease Activity.

[Figure: see text].

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.

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.

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.

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.

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

Purpose. To investigate how multipotent retinal stem cells (RSCs) isolated from mice respond to the proinflammatory signaling molecules, IL-1β, IL-18, and IL-17A. Materials and Methods.

The role of sex in uveitis and ocular inflammation.

Uveitides can be due to non-infectious and infectious etiologies. It has been observed that there is a gender difference with a greater preponderance of non-infectious uveitis in women than in men. This review will describe both non-infectious and infectious uveitides and describes some of the current autoimmune mechanisms thought to be underlying the gender difference.

Evaluating Potential Therapies in a Mouse Model of Focal Retinal Degeneration with Age-related Macular Degeneration (AMD)-Like Lesions.

Although the mouse has no macula leutea, its neuroretina and retinal pigment epithelium (RPE) can develop lesions mimicking certain features of age-related macular degeneration (AMD). Differences between the and double deficient mouse on () background (DKO ) and the mouse in photoreceptor and RPE pathology, as well as ocularA2E contents and immune responses, show that DKO recapitulates some human AMD-like features in addition to retinal dystrophy/degeneration. Different therapeutic interventions have been demonstrated to be effective on the AMD-like features of DKO mice.

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.