Inference of CRISPR Edits from Sanger Trace Data.

Efficient and precise genome editing requires a fast, quantitative, and inexpensive assay to assess genotype following editing. Here, we present ICE (Inference of CRISPR Edits), which enables robust analysis of CRISPR edits using Sanger data. ICE proposes potential outcomes for editing with guide RNAs, and then determines which are supported by the data via regression. The ICE algorithm is robust and reproducible, and it can be used to analyze CRISPR experiments within days after transfection. We also confirm that ICE produces accurate estimates of editing outcomes across a variety of benchmarks, and within the context of other existing Sanger analysis tools. The ICE tool is free to use and open source, and offers several improvements over current analysis tools, such as batch analysis and support for a variety of editing conditions. It is available online at ice.synthego.com, and the source code is available at github.com/synthego-open/ice.

Designing Anti-Microbial Peptides Against Major ß-Lactamase Enzymes in Clinically Important Gram-Negative Bacterial Pathogens: An In-Silico Study.

Anti-microbial resistance (AMR) creating healthcare concerns worldwide requires ardent exploration of therapeutic alternatives. Although anti-microbial peptides (AMP) are popular for broad-spectrum activity, recent evidence of increasing resistance to membrane-acting AMPs by ESKAPE pathogens has compelled us to design novel AMPs as therapeutic candidates. A library of 60 AMPs comprising natural AMPs and their mutants was constructed through in-silico methods. After physico-chemical property evaluations, each peptide in the library was subjected to flexible molecular docking against four major ß-lactamases in Gram-negative ESKAPE pathogens. Among the potent AMP mutants, a Lactoferricin B-Mutant (M4) possessed uniformly high affinity with SHV1, OXA48, NDM1, and AmpC having energies -842.0Kcal/mol, -774.8Kcal/mol, -1103.3Kcal/mol, and -858.8Kcal/mol respectively. Coarse-grained clustering and flexibility analysis further accounted for the residue-level stable configurations of the protein-peptide complexes with high affinity. Highest affinity of Lactoferricin B_M4 was found with NDM1 due to H-bonds, salt-bridges, and hydrophobic interactions with the metallo-ß-lactamase domain including crucial active-site residue Asp124. Molecular dynamics simulation further confirmed the stability of Lactoferricin B_M4-NDM1 complex having low residue-level root-mean square deviations (RMSD), atomic-level fluctuations, and radius of gyration (Rg). The study encourages experimental validations and similar methods to identify potential AMPs against drug-resistant pathogens.

CRISPRoff enables spatio-temporal control of CRISPR editing.

Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.

Abnormal preprocedural international normalized ratio and platelet counts are not associated with increased bleeding complications after ultrasound-guided thoracentesis.

OBJECTIVE: The objective of our study was to identify differences in hemorrhagic complications after ultrasound-guided thoracentesis on the basis of patient coagulation parameters. MATERIALS AND METHODS: The records of consecutive patients who underwent ultrasound-guided thoracentesis between January 1, 2008 and April 30, 2010 were reviewed to document the international normalized ratio (INR) and platelet count obtained within 72 hours before thoracentesis and to identify bleeding complications that occurred after the procedure. The observed complication rates and 95% CIs for differences in complication rates were calculated. RESULTS: There were 1076 procedures performed during the study period with no hemorrhagic complications identified (0% complication rate; 95% CI, 0.00-0.34%). INR values before thoracentesis were available for 822 procedures: INR exceeded 2.0 in 139 cases (17%), 2.5 in 59 cases (7%), and 3.0 in 32 cases (4%). The 95% CI for the 0% difference in complications observed between two groups of patients determined by specific INR values was -0.008 to 0.014 (INR, 1.5), -0.007 to 0.026 (INR, 2.0), -0.007 to 0.061 (INR, 2.5), and -0.009 to 0.11 (INR, 3.0). Platelet values before thoracentesis were available for 953 procedures; the platelet count was less than 100,000/µL for 148 procedures (16%), less than 50,000/µL for 58 procedures (6%), and less than 25,000/µL for 12 procedures (1%). The 95% CI for the 0% difference in complications between two groups of patients determined by a platelet count threshold of 50,000/µL was -0.007 to 0.062. CONCLUSION: The risk of bleeding after ultrasound-guided thoracentesis performed by radiologists is low even if the preprocedural INR and platelet count are abnormal. An approach in which no coagulation testing or correction is performed before thoracentesis may be justified.