The human genome produces thousands of long non-coding RNAs (lncRNAs) – transcripts >200 nucleotides long that do not encode proteins. While critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. Here, we developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in 7 diverse cell lines including 6 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.

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 CRISPR 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, and to dissect developmental pathways and model disease.

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 . Electronic supplementary material The online version of this article (10.1186/s13059-019-1783-3) contains supplementary material, which is available to authorized users.

15The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing 16 by even a single base pair. However, current tools only design sgRNAs for a reference genome, not taking 17 into account individual variants which may generate, remove, or modify CRISPR/Cas sgRNA sites. This 18 may cause mismatches between designed sgRNAs and the individual genome they are intended to target, 19 leading to decreased experimental performance. Here we describe AlleleAnalyzer, a tool for designing 20 personalized and allele-specific sgRNAs for genome editing. We leverage >2,500 human genomes to 21 identify optimized pairs of sgRNAs that can be used for human therapeutic editing in large populations in 22 the future. 23 24 25 2 Keywords 26 27 CRISPR, sgRNA design, genomics, genome surgery, genome editing, computational biology 28 Background 29The CRISPR/Cas genome-editing system is highly specific, with the ability to discriminate between similar 30 genomic sites, even alleles, based on a single nucleotide difference [1]. In order to target a genomic region 31 with the CRISPR system, a single-guide RNA (sgRNA) must be designed that is specific to the region of 32 interest. While current sgRNA design tools incorporate various data relating to predicted efficiency and 33 specificity such as epigenetic marks and chromatin accessibility [2][3][4], in the vast majority of cases, sgRNAs 34 are designed using reference genomes, such as the hg38 assembly for human or the GRCm38 assembly for 35 mouse. Since sgRNAs are often used on cell lines or organisms with many nucleotide differences from the 36 reference (e.g., on average 0.1% of a human genome [5]). Despite the finding that sgRNAs can sometimes 37 tolerate a single basepair mismatch, these mismatches frequently negatively impact sgRNA efficiency and 38 render imprecise the results of specificity prediction [2, 6, 7]. Furthermore, the use of CRISPR to research 39 areas such as haploinsufficiency, genomic imprinting, and dominant negative diseases require allele-40 specific sgRNA design. To address these challenges, we developed AlleleAnalyzer, a software tool that 41 designs personalized and allele-specific sgRNAs for individual genomes, identifies pairs of sgRNAs to 42 generate excisions likely to block expression of a gene, and leverages patterns of shared variation from 43 >2,500 human genomes to design sgRNA pairs for that will have the greatest utility in a target population. 44 45 Results and Discussion 46 47 Incorporating genetic variation into sgRNA design enables personalized and allele-specific CRISPR All possible personalized sgRNAs for SpCas9, SaCas9 and cpf1 (Cas12a) in the region surrounding the 404 first exon of RHO WTC (Supplementary Figure 6). WTC has no homozygous variants in this region, thus 405 allele frequency and variant-related columns are blank. However, the sgRNAs are designed to avoid the 9 406 heterozygous variants that WTC has in this region. 407408 Supplementary Table 4

Objectives: Intussusception is a pediatric medical emergency that can be difficult to diagnose. Radiologyperformed ultrasound is the diagnostic study of choice but may lead to delays due to lack of availability. Point-ofcare 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, multiplechoice 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.