DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA

To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.

tracks

tracks January 12-February 8, 1999 Jaffe-Friede & Strauss Galleries, Hopkins Center, Dartmouth College jin soo kim artist-in-residencehttps://via.library.depaul.edu/oral_his_gallery/1086/thumbnail.jp

Alcoholism and Diabetes Mellitus

Chronic use of alcohol is considered to be a potential risk factor for the incidence of type 2 diabetes mellitus (T2DM), which causes insulin resistance and pancreatic β-cell dysfunction that is a prerequisite for the development of diabetes. However, alcohol consumption in diabetes has been controversial and more detailed information on the diabetogenic impact of alcohol seems warranted. Diabetes, especially T2DM, causes dysregulation of various metabolic processes, which includes a defect in the insulin-mediated glucose function of adipocytes, and an impaired insulin action in the liver. In addition, neurobiological profiles of alcoholism are linked to the effects of a disruption of glucose homeostasis and of insulin resistance, which are affected by altered appetite that regulates the peptides and neurotrophic factors. Since conditions, which precede the onset of diabetes that are associated with alcoholism is one of the crucial public problems, researches in efforts to prevent and treat diabetes with alcohol dependence, receives special clinical interest. Therefore, the purpose of this mini-review is to provide the recent progress and current theories in the interplay between alcoholism and diabetes. Further, the purpose of this study also includes summarizing the pathophysiological mechanisms in the neurobiology of alcoholism

Risk factors for delayed and non-union following transfibular ankle arthrodesis

Background: This study was to identify risk factors associated with delayed union and non-union in patients who underwent transfibular ankle arthrodesis.Methods: This study included 43 patients who underwent ankle arthrodesis using transfibular approach between January 2012 and September 2018 and were followed up for more than 12 months. The patients were divided into two groups according to delayed union or non-union. Group A included patients who had delayed union or non-union and Group B included patients without these complications. Variables that could contribute to non-union including etiologies, age, chronic renal failure, hypertension, diabetes, smoking, pre-operative talus bone quality, pre-operative angulation of the talus and fixation methods were evaluated.Results: The mean time to bone union was 12.7±7.25 weeks. Group A included 12 patients with 5 cases of non-union and 7 cases of delayed union and group B included 31 patients. Infection of the ankle joint (OR, 1.73; p=0.041) was risk factor for non-union and delayed union on the basis of multivariate analysis.Conclusions: We concluded that infection of the ankle joint is the most significant risk factor for delayed union and nonunion in our study. Careful attention should be paid preoperatively, intraoperatively and postoperatively to patients who have this risk factor to obtain a satisfactory surgical outcome

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.