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CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression.IMPORTANCEToxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species.
Pubmed ID: 31243081
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A collection of tools for flow cytometer and application setup, data acquisition, and data analysis that help streamline flow cytometry workflows. It provides features to help users integrate flow systems into new application areas, including index sorting for stem cell and single-cell applications, as well as automation protocols for high-throughput and robotic laboratories.
View all literature mentionsGlobal nonprofit biological resource center (BRC) and research organization that provides biological products, technical services and educational programs to private industry, government and academic organizations. Its mission is to acquire, authenticate, preserve, develop and distribute biological materials, information, technology, intellectual property and standards for the advancement and application of scientific knowledge. The primary purpose of ATCC is to use its resources and experience as a BRC to become the world leader in standard biological reference materials management, intellectual property resource management and translational research as applied to biomaterial development, standardization and certification. ATCC characterizes cell lines, bacteria, viruses, fungi and protozoa, as well as develops and evaluates assays and techniques for validating research resources and preserving and distributing biological materials to the public and private sector research communities.
View all literature mentionsNon-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsNIH genetic sequence database that provides annotated collection of all publicly available DNA sequences for almost 280 000 formally described species (Jan 2014) .These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole-genome shotgun (WGS) and environmental sampling projects. Most submissions are made using web-based BankIt or standalone Sequin programs, and GenBank staff assigns accession numbers upon data receipt. It is part of International Nucleotide Sequence Database Collaboration and daily data exchange with European Nucleotide Archive (ENA) and DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through NCBI Entrez retrieval system, which integrates data from major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of GenBank database are available by FTP.
View all literature mentionsCommercial antibody vendor which supplies antibodies and other products to life science researchers.
View all literature mentionsCommercial vendor and service provider of laboratory reagents and antibodies. Supplier of scientific instrumentation, reagents and consumables, and software services.
View all literature mentionsSoftware for single-cell flow cytometry analysis. Its functions include management, display, manipulation, analysis and publication of the data stream produced by flow and mass cytometers.
View all literature mentionsAmerican chemical, life science and biotechnology company owned by Merck KGaA. Merger of Sigma Chemical Company and Aldrich Chemical Company. Provides organic and inorganic chemicals, building blocks, reagents, advanced materials and stable isotopes for chemical synthesis, medicinal chemistry and materials science, antibiotics, buffers, carbohydrates, enzymes, forensic tools, hematology and histology, nucleotides, proteins, peptides, amino acids and their derivatives.
View all literature mentionsAn approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing.
View all literature mentionsSoftware for image processing, analysis, and editing. The software includes features such as touch capabilities, a customizable toolbar, 2D and 3D image merging, and Cloud access and options.
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