Target-Specific Precision of CRISPR-Mediated Genome Editing.

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.

Evaluation of upper airway patency during Cheyne-Stokes breathing in heart failure patients.

Little is known about the changes in upper airway calibre in Cheyne-Stokes respiration (CSR) during sleep in patients with congestive heart failure. This study aimed to test the hypothesis that upper airway closure occurs during central CSR events, by assessing upper airway calibre during sleep using the forced oscillation technique (FOT). Nine males with compensated heart failure (left ventricular ejection fraction mean ± sem 27.9 ± 5.1%) and predominant central CSR (apnoea/hypopnoea index 43.9 ± 4.2 events · h(-1)) were studied during overnight polysomnography, which included pneumotachography, inductance plethysmography or oesophageal pressure and FOT-derived impedance signal (|Z|). Baseline |Z| values during stable breathing in stage 2 sleep were 11.0 ± 1.3 cmH(2)O · s · L(-1). Mean |Z| increased to 31.9 ± 6.7 cmH(2)O · s · L(-1) during obstructive apnoeas (7% of events, n = 46). Increases in |Z| consistent with upper airway narrowing (more than two-fold baseline) were common during central apnoeas (50 ± 12% of events) occurring in the middle or end of apnoeas and occurred during some central hypopnoeas (16 ± 10% of events), typically in the expiratory phase. These findings indicate that in heart failure patients, reductions in upper airway calibre are common during CSR apnoeas, and may also occur during central hypopnoeas.

High Q light-emitting Si-rich Si3N4 microdisks.

We report on the optical properties of active silicon (Si)-rich Si3N4 microdisk cavities in the visible range. We have studied the correlation between the quality (Q) factor of the cavities and the active material deposition parameters. Microphotoluminescence measurements revealed subangstrom whispering galley modes resonances and a maximum Q of 104 around 760 nm. These values improve significantly the best results reported so far for Si-based light-emitting circular resonators in the visible range. In contrast to what is reported for Si-rich SiO2-based microcavities, we demonstrate the absence of a spectral widening at high pump fluxes associated to carrier absorption mechanisms, which allows high emitted power without degrading the Q of the cavity. These results open the route toward the monolithic integration of those structures into more complex circuits including Si photodetectors.

Disruption of the MSL complex inhibits tumour maintenance by exacerbating chromosomal instability.

Rewiring of cellular programmes in malignant cells generates cancer-specific vulnerabilities. Here, using an unbiased screening strategy aimed at identifying non-essential genes required by tumour cells to sustain unlimited proliferative capacity, we identify the male-specific lethal (MSL) acetyltransferase complex as a vulnerability of genetically unstable cancers. We find that disruption of the MSL complex and consequent loss of the associated H4K16ac mark do not substantially alter transcriptional programmes but compromise chromosome integrity and promote chromosomal instability (CIN) that progressively exhausts the proliferative potential of cancer cells through a p53-independent mechanism. This effect is dependent on pre-existing genomic instability, and normal cells are insensitive to MSL disruption. Using cell- and patient-derived xenografts from multiple cancer types, we show that excessive CIN induced by MSL disruption inhibits tumour maintenance. Our findings suggest that targeting MSL may be a valuable means to increase CIN beyond the level tolerated by cancer cells without inducing severe adverse effects in normal tissues.

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays.

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions.