The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

Mixed reality, a blending of the physical and digital worlds, can enhance the surgical experience, leading to greater precision, efficiency, and improved outcomes. Various studies across different disciplines have reported encouraging results using mixed reality technologies, such as augmented and virtual reality. To provide a better understanding of the applications and limitations of this technology in plastic surgery, we performed a systematic review of the literature in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The initial query of the National Center for Biotechnology Information database yielded 2544 results, and only 46 articles met our inclusion criteria. The majority of studies were in the field of craniofacial surgery, and uses of mixed reality included preoperative planning, intraoperative guides, and education of surgical trainees. A deeper understanding of mixed reality technologies may promote its integration and also help inspire new and creative applications in healthcare.

OBJECTIVEThe 6-minute walking test (6WT) is used to determine restrictions in a subject’s 6-minute walking distance (6WD) due to lumbar degenerative disc disease. To facilitate simple and convenient patient self-measurement, a free and reliable smartphone app using Global Positioning System coordinates was previously designed. The authors aimed to determine normative values for app-based 6WD measurements.METHODSThe maximum 6WD was determined three times using app-based measurement in a sample of 330 volunteers without previous spine surgery or current spine-related disability, recruited at 8 centers in 5 countries (mean subject age 44.2 years, range 16–91 years; 48.5% male; mean BMI 24.6 kg/m2, range 16.3–40.2 kg/m2; 67.9% working; 14.2% smokers). Subjects provided basic demographic information, including comorbidities and patient-reported outcome measures (PROMs): visual analog scale (VAS) for both low-back and lower-extremity pain, Core Outcome Measures Index (COMI), Zurich Claudication Questionnaire (ZCQ), and subjective walking distance and duration. The authors determined the test-retest reliability across three measurements (intraclass correlation coefficient [ICC], standard error of measurement [SEM], and mean 6WD [95% CI]) stratified for age and sex, and content validity (linear regression coefficients) between 6WD and PROMs.RESULTSThe ICC for repeated app-based 6WD measurements was 0.89 (95% CI 0.87–0.91, p < 0.001) and the SEM was 34 meters. The overall mean 6WD was 585.9 meters (95% CI 574.7–597.0 meters), with significant differences across age categories (p < 0.001). The 6WD was on average about 32 meters less in females (570.5 vs 602.2 meters, p = 0.005). There were linear correlations between average 6WD and VAS back pain, VAS leg pain, COMI Back and COMI subscores of pain intensity and disability, ZCQ symptom severity, ZCQ physical function, and ZCQ pain and neuroischemic symptoms subscores, as well as with subjective walking distance and duration, indicating that subjects with higher pain, higher disability, and lower subjective walking capacity had significantly lower 6WD (all p < 0.001).CONCLUSIONSThis study provides normative data for app-based 6WD measurements in a multicenter sample from 8 institutions and 5 countries. These values can now be used as reference to compare 6WT results and quantify objective functional impairment in patients with degenerative diseases of the spine using z-scores. The authors found a good to excellent test-retest reliability of the 6WT app, a low area of uncertainty, and high content validity of the average 6WD with commonly used PROMs.