Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

ObjectiveClinical use of CHADS2 and CHA2DS2-VASc scoring systems for predicting AF following cardiac surgery have been reported in previous studies and demonstrated well-validated predictive value. We sought to investigate whether the two scoring systems are effective for predicting new-onset of AF following cardiac valve surgery and to demonstrate its potential utility of clinical assessment.MethodsMedical records of all patients underwent cardiac valve surgeries during the period of January 2003 and December 2013 without preoperative AF at the cardiac center of our university were reviewed. The main outcome end point of our study was the early new-onset of AF following cardiac valve surgery.ResultsThere were overall 518 patients involved in this study, with 234 (45.17%) developed POAF following valve surgery. Patients with POAF had older age (P=0.23) and higher BMI (P=0.013) than those without POAF. History of heart failure (P=0.025), hypertension (P=0.021), previous stroke or TIA (P=0.032), coronary artery disease (P=0.001), carotid artery disease (P=0.024) and preoperative medication of statins (P=0.021) were significantly more recorded in POAF group. Patients with POAF also had higher LAD (P=0.013) and E/e’ ratio (P<0.001). The CHADS2 and CHA2DS2-VASc scores were significantly higher in patients with POAF (P=0.002; P<0.001), and under univariate and multivariate regression analysis the CHADS2 and CHA2DS2-VASc scores were significant predictors of POAF (P=0.001; P<0.001). Based on stratification of CHADS2 and CHA2DS2-VASc scores, the Kaplan-Meier analysis obtained a higher POAF rate on patients with higher stratification of CHADS2 and CHA2DS2-VASc scores (P<0.001; P<0.001).ConclusionIn conclusion, CHADS2 and CHA2DS2-VASc scores were directly associated with the incidence of POAF following valve surgery and a higher score was strongly predictive of POAF.

Diabetes mellitus (DM) facilitates atrial fibrosis and increases the risk of atrial fibrillation (AF). The underlying mechanism of DM in causing AF remains mostly unknown and potential therapeutic targets for DM-induced AF are rarely reported. Hydrogen sulfide (H 2 S) has drawn considerable attention in recent years for its potential as a cardiovascular protector. Thus, the aim of the present study was to investigate the effect of H 2 S on DM-induced AF and the mechanism of action. Sprague-Dawley rats were divided into four groups, including the control group, the DM group, the H 2 S group and the DM+H 2 S group. The DM group and the DM+H 2 S group were administered streptozotocin to induce DM, whereas the other two groups were given citrate buffer as a control. The H 2 S group and the DM+H 2 S group were administered with an intraperitoneal injection of sodium hydrosulfide (precursor of H 2 S). AF inducibility, AF duration, atrial fibrosis and vital protein expression of oxidative stress were compared among the four groups. The DM group showed significantly higher AF incidence rates and duration (P<0.05). Histology results demonstrated severe atrial fibrosis in the DM group, and the PI3K/Akt/endothelial nitric oxide synthase (eNOS) pathway was significantly downregulated (P<0.05). However, when H 2 S was administered, the rats showed lower AF incidence and duration compared with the DM group. Additionally, H 2 S was able to mitigate the atrial fibrosis induced by DM, as well as the proliferation and migration of cardiac fibroblasts, as demonstrated by an MTT assay and real-time cell analyzer migration experiment. Western blotting showed that the expression levels of the PI3K/Akt/eNOS pathway in the DM+H 2 S group were significantly upregulated compared with those of the DM group (P<0.05). In summary, DM status can lead to the structural remodeling of atrial fibrosis, facilitating AF incidence and persistence. Administration of H 2 S does not affect the glucose level, but can significantly mitigate atrial fibrosis and reduce the incidence of AF induced by DM, probably via activation of the PI3K/Akt/eNOS pathway.

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