CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.

No abstract

No abstract

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5'-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1-10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.

No abstract