ABSTRACT
Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.
Subject(s)
Flow Cytometry/methods , High-Throughput Screening Assays/methods , Inflammation/genetics , Inflammation/metabolism , Macrophages/physiology , NF-kappa B/physiology , Tumor Necrosis Factor-alpha/physiology , 3' Untranslated Regions , Animals , CRISPR-Cas Systems , Cell Line , Cell Survival , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Expression Regulation , HEK293 Cells , Humans , Mice , RNA, Guide, Kinetoplastida/genetics , Signal TransductionABSTRACT
BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells. RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA. CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Genetic Testing/methods , Molecular Typing/methods , RNA, Guide, Kinetoplastida , Humans , Jurkat CellsABSTRACT
Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention.
Subject(s)
CRISPR-Associated Protein 9/genetics , Epistasis, Genetic/genetics , Gene Regulatory Networks/genetics , Computational Biology , Gene Knockout Techniques , Humans , K562 Cells , Staphylococcus aureus/genetics , Streptococcus pyogenes/genetics , Transcriptional Activation/geneticsABSTRACT
A 64-channel brain array coil was developed and compared to a 32-channel array constructed with the same coil former geometry to precisely isolate the benefit of the 2-fold increase in array coil elements. The constructed coils were developed for a standard clinical 3T MRI scanner and used a contoured head-shaped curved former around the occipital pole and tapered in at the neck to both improve sensitivity and patient comfort. Additionally, the design is a compact, split-former design intended for robust daily use. Signal-to-noise ratio and noise amplification (G-factor) for parallel imaging were quantitatively evaluated in human imaging and compared to a size and shape-matched 32-channel array coil. For unaccelerated imaging, the 64-channel array provided similar signal-to-noise ratio in the brain center to the 32-channel array and 1.3-fold more signal-to-noise ratio in the brain cortex. Reduced noise amplification during highly parallel imaging of the 64-channel array provided the ability to accelerate at approximately one unit higher at a given noise amplification compared to the sized-matched 32-channel array. For example, with a 4-fold acceleration rate, the central brain and cortical signal-to-noise ratio of the 64-channel array was 1.2- and 1.4-fold higher, respectively, compared to the 32-channel array. The characteristics of the coil are demonstrated in accelerated brain imaging.
