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Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii

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In Vitro Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1498))

Abstract

Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.

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Acknowledgments

Partially supported by NIH grant AI118426, AI034036 (to LDS) and Projects 2662015PY048 and 2662015PY104 from the Fundamental Research Funds for the Central Universities (to B.S.). We thank Dr. Joshua B. Radke for a critical reading of the manuscript.

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    Authors and Affiliations

    1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, 430070, China

      Bang Shen

    2. Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO, 63110, USA

      Kevin Brown, Shaojun Long & L. David Sibley

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    1. Bang Shen
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    2. Kevin Brown
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    3. Shaojun Long
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    4. L. David Sibley
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    Correspondence to L. David Sibley .

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    1. Coskata, Inc., Warrenville, Illinois, USA

      Andrew Reeves

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    Shen, B., Brown, K., Long, S., Sibley, L.D. (2017). Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii . In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_6

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    • DOI: https://doi.org/10.1007/978-1-4939-6472-7_6

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