pooled-crispr-based-genetic-screens-in-mammalian-cells

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

University of Toronto

gamma-Hydroxybutyric acid (GHB) is a short-chain fatty acid that occurs naturally in mammalian brain where it is derived metabolically from gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the brain. GHB was synthesised over 40 years ago and its presence in the brain and a number of aspects of its biological, pharmacological and toxicological properties have been elucidated over the last 20-30 years. However, widespread interest in this compound has arisen only in the past 5-10 years, primarily as a result of the emergence of GHB as a major recreational drug and public health problem in the US. There is considerable evidence that GHB may be a neuromodulator in the brain. GHB has multiple neuronal mechanisms including activation of both the gamma-aminobutyric acid type B (GABA(B)) receptor, and a separate GHB-specific receptor. This complex GHB-GABA(B) receptor interaction is probably responsible for the protean pharmacological, electroencephalographic, behavioural and toxicological effects of GHB, as well as the perturbations of learning and memory associated with supra-physiological concentrations of GHB in the brain that result from the exogenous administration of this drug in the clinical context of GHB abuse, addiction and withdrawal. Investigation of the inborn error of metabolism succinic semialdehyde deficiency (SSADH) and the murine model of this disorder (SSADH knockout mice), in which GHB plays a major role, may help dissect out GHB- and GABA(B) receptor-mediated mechanisms. In particular, the mechanisms that are operative in the molecular pathogenesis of GHB addiction and withdrawal as well as the absence seizures observed in the GHB-treated animals.

Gamma-hydroxybutyric acid: neurobiology and toxicology of a recreational drug.

In this study, we tested whether over-expressing the GABA(B) receptor R1a subtype in transgenic mouse forebrain neurons would be sufficient to induce spontaneous absence seizures. As hypothesized, these transgenic mice develop spontaneous, recurrent, bilaterally synchronous, 3-6 Hz slow spike and wave discharges between 2 and 4 months of age. These discharges are blocked by ethosuximide and exacerbated by baclofen confirming their absence nature. The discharges occur coincident with absence-like behaviors such as staring, facial myoclonus, and whisker twitching. However, in contrast to typical absence epilepsy models, these mice move during the ictal event, display spike and wave discharges in both thalamocortical and limbic circuitry, exhibit impaired hippocampal synaptic plasticity, and display significantly impaired learning ability. Collectively, these features are more characteristic of the less common but more debilitating atypical form of absence epilepsy. Thus, these data support a role for the GABA(B)R1a receptor subtype in the etiology of atypical absence epilepsy.

Transgenic mice over-expressing GABA(B)R1a receptors acquire an atypical absence epilepsy-like phenotype.

Altered cerebrovascular blood flow has been proposed as a mechanism for multiple sclerosis (MS). The primary objective of this study was to measure arterial and venous blood flow in adolescent MS patients and healthy controls (HC), in whom confounding factors such as age and lifestyle are less influential.

Cerebral arterial and venous blood flow in adolescent multiple sclerosis patients and age-matched controls using phase contrast MRI.

Administering multiple intravenous (IV) infusions to a single patient via infusion pump occurs routinely in health care, but there has been little empirical research examining the risks associated with this practice or ways to mitigate those risks.

Multiple Intravenous Infusions Phase 2b: Laboratory Study.

Walking or locomotor training is often initiated following pediatric spinal cord injury (SCI). There is no synthesis of the literature on interventions targeting walking for pediatric SCI, although this would assist future clinical trials and interventions. To address this need, we completed a systematic review to summarize the who, what, when, and how of walking interventions in children with SCI. Participant characteristics, training parameters, and walking outcomes with training in pediatric SCI were identified and compared with training parameters and outcomes in adults with SCI. The PubMed, Medline, AMED, Embase, PsycInfo, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and CINAHL databases were searched for studies that included participants aged 1-17 years with a SCI acquired post-birth, physical interventions, and pre- and post-training walking measures. Two researchers evaluated each study's risk of bias using a domain-based approach. Training parameters and walking outcomes were extracted. Total training duration (duration × frequency × number of weeks) was calculated. Thirteen pediatric studies (n = 43 children) were included; all but one were case series/reports. Risk of bias was high in the pediatric studies. A 2012 adult review was updated (11 studies added). As with adults, the training durations, frequencies, and modes used with the children varied; however, overground walking practice was included in 10/13 pediatric studies. Improvements in walking capacity, speed, and distance were comparable between children and adults. There was a trend for greater gains with greater total training durations. There is a paucity of high-quality research examining interventions targeting walking after pediatric SCI; however, intensive training, including practice overground, results in notable improvements.

Training to Improve Walking after Pediatric Spinal Cord Injury: A Systematic Review of Parameters and Walking Outcomes.

Abstracts and Workshops 7th National Spinal Cord Injury Conference November 9 - 11, 2017 Fallsview Casino Resort Niagara Falls, Ontario, Canada.

CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.

Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

Initiatives to increase physical activity (PA) are most effective when combined with behavior change strategies (BCS). However, this is not routinely done and perhaps can be attributed to health care professionals' (HCP) lack of confidence and understanding of how to best apply BCS. This review aimed to identify and synthesize the existing literature describing methods used to train HCP to deliver BCS for PA promotion for individuals living with neurological conditions. This review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Eight databases were searched for articles describing how HCP were trained on BCS targeting PA and/or exercise for neurological pathology. Two researchers independently screened abstracts and used third-party consensus when needed. Studies must have described the planning/implementation of BCS targeting PA in adults with neurological disorders, with interventions delivered by a HCP. A data extraction table based on the study objective and the Cochrane Consumers and Communication Review Group's data extraction template was used. Risk of bias was assessed using the Quality Assessment Tools for Quantitative and Qualitative Studies. Included studies were also evaluated using the Workgroup for Intervention Development and Evaluation Research (WIDER) recommendations for reporting behavior change intervention content. Extraction and evaluation of risk of bias were completed independently by two researchers. Data were synthesized according to a who, what, when, and how approach. The search yielded 2,616 articles after duplicates removed, with 13 articles, representing eight unique interventions in individuals with spinal cord injury, stroke, multiple sclerosis, and Parkinson's disease included. Methodological quality ranged from strong to weak. Methods used to increase HCP knowledge and use of BCS were heterogeneous. Common training features included multiple resources, multiple sessions, expert input, and ongoing support, but a lack of evaluation of training effectiveness. The BCS were based on Social Cognitive Theory in seven interventions. Physiotherapists were the most common HCP trained. There was considerable variety in how HCP were trained to use BCS and a lack of evaluation of training effectiveness. Future work should include more description of the content and structure of HCP training and a direct measure of effectiveness. Researchers should consider implementation strategies during development and reporting of behavior change interventions. This review was not registered with PROSPERO.

Informing the training of health care professionals to implement behavior change strategies for physical activity promotion in neurorehabilitation: a systematic review.

Impaired reactive balance control can lead to increased falls in people with neurological impairments. Perturbation-based balance training (PBT), which involves repetitive exposure to destabilizing external perturbations, improves the ability to take reactive steps in older adults and individuals who have had a stroke.

Intensive Balance Training for Adults With Incomplete Spinal Cord Injuries: Protocol for an Assessor-Blinded Randomized Clinical Trial.

Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.

Article	Year
Gamma-hydroxybutyric acid: neurobiology and toxicology of a recreational drug. Toxicological reviews\| PubMed ID: 15298489	2004
Transgenic mice over-expressing GABA(B)R1a receptors acquire an atypical absence epilepsy-like phenotype. Neurobiology of disease\| PubMed ID: 17363260	2007
Cerebral arterial and venous blood flow in adolescent multiple sclerosis patients and age-matched controls using phase contrast MRI. Journal of magnetic resonance imaging : JMRI\| PubMed ID: 24115149	2014
Multiple Intravenous Infusions Phase 2b: Laboratory Study. Ontario health technology assessment series\| PubMed ID: 26316919	2014
Training to Improve Walking after Pediatric Spinal Cord Injury: A Systematic Review of Parameters and Walking Outcomes. Journal of neurotrauma\| PubMed ID: 27869534	2017
Abstracts and Workshops 7th National Spinal Cord Injury Conference November 9 - 11, 2017 Fallsview Casino Resort Niagara Falls, Ontario, Canada. The journal of spinal cord medicine\| PubMed ID: 29034821	2017
Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30324523	2019
Informing the training of health care professionals to implement behavior change strategies for physical activity promotion in neurorehabilitation: a systematic review. Translational behavioral medicine\| PubMed ID: 30496560	2018
Intensive Balance Training for Adults With Incomplete Spinal Cord Injuries: Protocol for an Assessor-Blinded Randomized Clinical Trial. Physical therapy\| PubMed ID: 30576475	2019
Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies. Cell reports\| PubMed ID: 30970261	2019
Reactive stepping after a forward fall in people living with incomplete spinal cord injury or disease. Spinal cord\| PubMed ID: 31358908	2019

Katherine Chan

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