Minimal genome-wide human CRISPR-Cas9 library.

CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.

Best practice for CRISPR design using current tools and resources.

Users facing the task of designing gRNAs for a CRISPR-mutagenesis experiment are typically confronted with a large variety of possible tools and existing libraries. Here we examine the design principles for such resources, and suggest a best practice which allows a user to evaluate and effectively use any of the existing CRISPR design tools or genome-wide libraries.

Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.

WGE: a CRISPR database for genome engineering.

UNLABELLED: The rapid development of CRISPR-Cas9 mediated genome editing techniques has given rise to a number of online and stand-alone tools to find and score CRISPR sites for whole genomes. Here we describe the Wellcome Trust Sanger Institute Genome Editing database (WGE), which uses novel methods to compute, visualize and select optimal CRISPR sites in a genome browser environment. The WGE database currently stores single and paired CRISPR sites and pre-calculated off-target information for CRISPRs located in the mouse and human exomes. Scoring and display of off-target sites is simple, and intuitive, and filters can be applied to identify high-quality CRISPR sites rapidly. WGE also provides a tool for the design and display of gene targeting vectors in the same genome browser, along with gene models, protein translation and variation tracks. WGE is open, extensible and can be set up to compute and present CRISPR sites for any genome. AVAILABILITY AND IMPLEMENTATION: The WGE database is freely available at www.sanger.ac.uk/htgt/wge CONTACT: : vvi@sanger.ac.uk or skarnes@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A new tool for converting food frequency questionnaire data into nutrient and food group values: FETA research methods and availability.

OBJECTIVES: To describe the research methods for the development of a new open source, cross-platform tool which processes data from the European Prospective Investigation into Cancer and Nutrition Norfolk Food Frequency Questionnaire (EPIC-Norfolk FFQ). A further aim was to compare nutrient and food group values derived from the current tool (FETA, FFQ EPIC Tool for Analysis) with the previously validated but less accessible tool, CAFÉ (Compositional Analyses from Frequency Estimates). The effect of text matching on intake data was also investigated. DESIGN: Cross-sectional analysis of a prospective cohort study-EPIC-Norfolk. SETTING: East England population (city of Norwich and its surrounding small towns and rural areas). PARTICIPANTS: Complete FFQ data from 11 250 men and 13 602 women (mean age 59 years; range 40-79 years). OUTCOME MEASURES: Nutrient and food group intakes derived from FETA and CAFÉ analyses of EPIC-Norfolk FFQ data. RESULTS: Nutrient outputs from FETA and CAFÉ were similar; mean (SD) energy intake from FETA was 9222 kJ (2633) in men, 8113 kJ (2296) in women, compared with CAFÉ intakes of 9175 kJ (2630) in men, 8091 kJ (2298) in women. The majority of differences resulted in one or less quintile change (98.7%). Only mean daily fruit and vegetable food group intakes were higher in women than in men (278 vs 212 and 284 vs 255 g, respectively). Quintile changes were evident for all nutrients, with the exception of alcohol, when text matching was not executed; however, only the cereals food group was affected. CONCLUSIONS: FETA produces similar nutrient and food group values to the previously validated CAFÉ but has the advantages of being open source, cross-platform and complete with a data-entry form directly compatible with the software. The tool will facilitate research using the EPIC-Norfolk FFQ, and can be customised for different study populations.

Dietary intake measurement using 7 d diet diaries in British men and women in the European Prospective Investigation into Cancer-Norfolk study: a focus on methodological issues.

The aim of the present study was to describe the energy, nutrient and crude v. disaggregated food intake measured using 7 d diet diaries (7dDD) for the full baseline Norfolk cohort recruited for the European Prospective Investigation into Cancer (EPIC-Norfolk) study, with emphasis on methodological issues. The first data collection took place between 1993 and 1998 in Norfolk, East Anglia (UK). Of the 30,445 men and women, aged 40-79 years, registered with a general practitioner invited to participate in the study, 25,639 came for a health examination and were asked to complete a 7dDD. Data from diaries with data recorded for at least 1 d were obtained for 99% members of the cohort; 10,354 (89·8%) of the men and 12,779 (91·5%) of the women completed the diet diaries for all 7 d. Mean energy intake (EI) was 9·44 (SD 2·22) MJ/d and 7·15 (SD 1·66) MJ/d, respectively. EI remained approximately stable across the days, but there was apparent under-reporting among the participants, especially among those with BMI >25 kg/m². Micronutrient density was higher among women than among men. In conclusion, under-reporting is an issue, but not more so than that found in national surveys. How foods were grouped (crude or disaggregated) made a difference to the estimates obtained, and comparison of intakes showed wide limits of agreement. The choice of variables influences estimates obtained from the food group data; while this may not alter the ranking of individuals within studies, this issue may be relevant when comparing absolute food intakes between studies.