Evaluating a Web-based Point-of-care Ultrasound Curriculum for the Diagnosis of Intussusception.

OBJECTIVES: Intussusception is a pediatric medical emergency that can be difficult to diagnose. Radiology-performed ultrasound is the diagnostic study of choice but may lead to delays due to lack of availability. Point-of-care ultrasound for intussusception (POCUS-I) studies have shown excellent accuracy and reduced lengths of stay, but there are limited POCUS-I training materials for pediatric emergency medicine (PEM) providers. METHODS: We performed a prospective cohort study assessing PEM physicians undergoing a primarily Web-based POCUS-I curriculum. We developed the POCUS-I curriculum using Kern's six-step model. The curriculum included a Web-based module and a brief, hands-on practice that was developed with a board-certified pediatric radiologist. POCUS-I technical skill, knowledge, and confidence were determined by a direct observation checklist, multiple-choice test, and a self-reported Likert-scale survey, respectively. We assessed participants immediately pre- and postcourse as well as 3 months later to assess for retention of skill, knowledge, and confidence. RESULTS: A total of 17 of 17 eligible PEM physicians at a single institution participated in the study. For the direct observation skills test, participants scored well after the course with a median (interquartile range [IQR]) score of 20 of 22 (20-21) and maintained high scores even after 3 months (20 [20-21]). On the written knowledge test, there was significant improvement from 57.4% (95% CI = 49.8 to 65.2) to 75.3% (95% CI = 68.1 to 81.6; p < 0.001) and this improvement was maintained at 3 months at 81.2% (95% CI = 74.5 to 86.8). Physicians also demonstrated improved confidence with POCUS-I after exposure to the curriculum, with 5.9% reporting somewhat or very confident prior to the course to 76.5% both after the course and after 3 months (p < 0.001). CONCLUSION: After a primarily Web-based curriculum for POCUS-I, PEM physicians performed well in technical skill in POCUS-I and showed improvement in knowledge and confidence, all of which were maintained over 3 months.

AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design.

The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at https://github.com/keoughkath/AlleleAnalyzer .

CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells.

The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.