Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq.

Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.

Povidone-iodine in vitro antiseptic efficacy as a function of exposure duration, concentration, preparation, and length of storage.

PURPOSE: Although 5% povidone-iodine (PVP-I) is frequently used as an ocular antiseptic agent, there is a lack of consensus regarding the effects of PVP-I concentration, storage after opening, and compounded preparation on PVP-I antisepsis. We performed a series of in-vitro experiments to determine the impact of these factors on PVP-I's inhibition of common causes of post-procedural eye infection. METHODS: Inhibition of microorganism growth was measured in-vitro as a function of active PVP-I exposure time. In control experiments, PVP-I was inactivated before microorganism exposure. Tested PVP-I solutions varied in concentration (0.6%, 5%, or 10%), length of storage after opening (0, 7, or 30 days), and preparation (commercial vs.compounded from stock PI solution). Tested pathogens included S. epidermidis, S. viridans, P. aeruginosa, methicillin-resistant S. aureus, methicillin-sensitive S. aureus, and C. albicans. RESULTS: PVP-I solutions inhibited all bacterial growth by 3 min and fungal growth by 15 s. Compared to 5% PVP-I, the 0.6% PVP-I was less effective in inhibiting S. viridans growth (200 ± 0 colonies vs. 7 ± 8 at 30 s, P = 0.0004; 183 ± 21 vs. 0 ± 0 at 1 min, P = 0.018), but more effective in inhibiting P. aeruginosa (30 ± 20 vs. 200 ± 0 at 15 s, P = 0.019). Compared to commercial and newly-opened PVP-I solutions, compounded preparations and solutions stored for 7 or 30 days after bottle opening either preserved or improved antiseptic efficacy against tested microorganisms. CONCLUSIONS: Concentration of PVP-I solution affects antiseptic efficacy within 1 min of exposure, but all solutions performed equivalently at 3 min. In contrast to results of prior studies investigating dilute PVP-I, the 0.6% PVP-I did not demonstrate a uniformly equivalent or superior anti-septic effect. Compounded preparation and storage length after bottle opening did not decrease PVP-I antiseptic activity.

Comparing Ahmed-FP7 to Baerveldt-250 and Baerveldt-350 surgical outcomes: 1-year results from a retrospective cohort study leveraging the electronic health record.

OBJECTIVE: To compare outcomes following Ahmed-FP7 (AGI-FP7), Baerveldt-250mm2 (BGI-250), or Baerveldt-350mm2 (BGI-350) implantation. METHODS AND ANALYSIS: Retrospective cohort study comprising 800 eyes from 800 individuals who underwent surgery 1 January 2016-31 December 2020 at a tertiary-care institution. Data were extracted from standardised fields in the electronic health record. Primary outcome was failure (defined as intraocular pressure (IOP) ≤5 mm Hg or >18 mm Hg or reduction <20% at two consecutive visits from month 3 onwards; or visual acuity (VA) loss ≥3 lines; or return to the operating room (OR)). Secondary outcomes were IOP, VA, number of follow-up visits and return to the OR. RESULTS: A total of 523 AGI-FP7, 133 BGI-250 and 144 BGI-350 cases were analysed. The AGI-FP7 group was more likely to be younger and diagnosed with secondary glaucoma, with a higher mean baseline IOP (28.5±12.2 vs 22.0±7.7 mm Hg in BGI-250 and 23.4±9.0 in BGI-350, p<0.001). Cumulative failure rate at month 12 was 30% (AGI-FP7) vs 39% (BGI-250) vs 33% (BGI-350, p=0.159). Mean IOP at month 12 was lower in the BGI-350 group compared with AGI-FP7 (12.4±4.4 vs 14.8±5.6 mm Hg, p=0.003) but not BGI-250 (vs 13.1±4.6, p=0.710). Target IOP was achieved in 71% of AGI-FP7, 66% BGI-250, and 76% BGI-350. VA loss and rates of return to the OR did not differ between groups. Both BGI-250 and BGI-350 had more follow-up visits than AGI-FP7 (p<0.001). CONCLUSION: These three glaucoma drainage devices performed similarly within 1 year, with no difference in failure rates despite differing baseline patient characteristics.

Otolith Membrane Herniation, not Semicircular Canal Duct Dilation, Is Associated with Decreased Caloric Responses in Ménière's Disease.

Ménière's disease (MD) is a debilitating disorder with unclear pathophysiology whose diagnosis often relies on clinical judgment rather than objective testing. To complicate matters further, a dissociation has emerged between two vestibular function tests commonly used in patients with MD to examine the same end-organ (the semicircular canals): the caloric test and video head impulse testing (vHIT). Caloric responses are often abnormal, while vHIT results remain normal. Explaining this dissociation could reveal novel insights into MD pathophysiology. Here, we conduct a histopathological study using temporal bone specimens (N = 58, 21 MD-affected ears and 37 age-matched controls) and their clinical testing data to examine current hypotheses aimed at this dissociation. We find otolith membrane herniation into the horizontal semicircular canal in 69% of MD ears, with 90% of these ears demonstrating a diminished caloric response. No ears with a normal response had this herniation. Moreover, we evaluated the semicircular canals for endolymphatic hydrops, which had been hypothesized to contribute to the dissociation, and found no evidence of duct dilation/hydrops. We did, however, note a potentially novel morphologic finding-smaller bony labyrinth cross-sectional diameters/areas in some MD ear canals compared to controls, suggesting relative size of the membranous duct to the bony canal rather than absolute size may be of importance. Taken together, this study refines hypotheses on the vestibular test dissociation in MD, holding diagnostic implications and expanding our understanding of the mechanisms underlying this enigmatic disease.