Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting.

BACKGROUND: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. RESULTS: Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. CONCLUSIONS: CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.

Preliminary Experience With Quadratus Lumborum Catheters for Postoperative Pain Management in Pediatric-Aged Patients With Contraindications to Epidural Anesthesia.

Background: Although neuraxial techniques such as caudal and epidural anesthesia were initially the predominant regional anesthetic technique used to provide postoperative analgesia in children, there has been a transition to the use of peripheral nerve blockade such as the quadratus lumborum block (QLB). We present preliminary experience with QL catheters for continuous postoperative analgesia in a cohort of pediatric patients following colorectal surgery. Methods: After institutional review board (IRB) approval, we retrospectively reviewed the records of patients who underwent major colorectal surgery and received QL catheters for postoperative analgesia. The postoperative pain control data consisted of QL catheter characteristics, anesthetic agents, adjuncts, pain scores, and opioid consumption during the postoperative period. Results: The study cohort included eight pediatric patients, ranging in age from 1 to 19 years (median age 11.8 years). The QL catheters were placed in the operating room after the induction of anesthesia. Comorbid conditions in the cohort that were contraindications to neuraxial anesthesia included spinal/vertebral malformations, presence of a ventriculoperitoneal (VP) shunt, anal atresia, tracheo-esophageal fistula (VACTERL) association, and coagulation disturbances. All patients underwent complex colorectal or genito-urologic procedures. Bilateral QL catheters were placed in six patients, and unilateral catheters were placed in two patients. Four patients received 0.5% ropivacaine and four patients received 0.2% ropivacaine of an initial bolus. The local anesthetic used for continuous infusion was 0.2% ropivacaine in five patients, 0.1% ropivacaine in two patients, and 1.5% chloroprocaine in one patient, with a median infusion rate of 0.11 mL/kg/h. QL catheter infusions were supplemented with intravenous opioids delivered by patient-controlled or nurse-controlled analgesia. The median opioid requirements in oral morphine milligram equivalents (MME) were 1.2, 1.0, 1.1, 0.5, and 0.6 MME/kg on postoperative days 1 - 5. Daily median pain scores were ≤ 2 during the 5-day postoperative course. All catheters functioned successfully and were in place for a median of 79.3 h. Other than early inadvertent removal of two catheters, no adverse effects were noted. Conclusions: Although our preliminary data suggest the efficacy of QL catheters in providing prolonged postoperative analgesia for up to 3 - 5 days following colorectal procedures, attention needs to be directed at measures to ensure that the catheter is secured to avoid inadvertent removal.

CrystalGrower: a generic computer program for Monte Carlo modelling of crystal growth.

A Monte Carlo crystal growth simulation tool, CrystalGrower, is described which is able to simultaneously model both the crystal habit and nanoscopic surface topography of any crystal structure under conditions of variable supersaturation or at equilibrium. This tool has been developed in order to permit the rapid simulation of crystal surface maps generated by scanning probe microscopies in combination with overall crystal habit. As the simulation is based upon a coarse graining at the nanoscopic level features such as crystal rounding at low supersaturation or undersaturation conditions are also faithfully reproduced. CrystalGrower permits the incorporation of screw dislocations with arbitrary Burgers vectors and also the investigation of internal point defects in crystals. The effect of growth modifiers can be addressed by selective poisoning of specific growth sites. The tool is designed for those interested in understanding and controlling the outcome of crystal growth through a deeper comprehension of the key controlling experimental parameters.