Eukaryotic-driven directed evolution of Cas9 nucleases.

BACKGROUND: Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity. RESULTS: Here, we develop a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. CONCLUSIONS: We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

Micropulse Laser Trabeculoplasty with 577 nm Wavelength at 1500 or 1000 mW for Primary Open-Angle Glaucoma: A Pilot Study.

Aim: To investigate the efficacy and safety of micropulse laser trabeculoplasty (MLT) using a 577 nm yellow wavelength laser randomly assigned to either 1500 or 1000 mW in patients with primary open-angle glaucoma (POAG). Methods: A prospective, double-blinded study of POAG patients was performed in a single center. MLT treatment included a 577 nm micropulse laser (IRIDEX IQ 577TM, IRIDEX, Mountain View, CA, USA) to 360° of the trabecular meshwork at randomly assigned varying powers: 1500 mW in one eye (MLT 1500 group) and 1000 mW in the other (MLT 1000 group). Best-corrected visual acuity (BCVA), intraocular pressure (IOP), corneal central thickness (CCT), and endothelial cell count (ECC) were evaluated at baseline (T0), post-operative 1 h (T1), 24 h (T2), 1 month (T3), 3 months (T4), and 6 months (T5) after laser treatment. Topical medications were assessed pre-treatment and at T4. Results: Among the 18 eyes included, we achieved a success rate (IOP reduced > 20%) in 77% of sampled eyes. In particular, IOP reduced at T2 and T3 with both MLT 1500 and 1000 without any significant differences (IOP reduction 22.9% vs. 17.3%, respectively, MLT1500 vs. MLT1000 at T2). The IOP returned to baseline values at T4 and T5 in both groups, with a reduction in topical medications administered from 2.5 ± 1.1 to 2.0 ± 1.2 to the 1500 mW group and from 2.4 ± 1.0 to 1.9 ± 1.0 to the 1000 mW group. At 1 h post-laser treatment, a transient IOP spike was registered among the MLT1500 group. There were no differences in CCT and ECC at any timepoint according to the laser powers. Conclusions: Over a 6-month follow-up period, 577 nm MLT at either 1500 or 1000 mW reduces IOP, enabling a stable reduction in the number of topical medications required for patients treated for POAG without any significant difference in terms of effectiveness and safety.