Development of CRISPR/cas9 for efficient genome editing in toxoplasma gondii

Bang Shen, Kevin Brown, Shaojun Long, L. David Sibley

Research output: Contribution to journalArticle

25 Scopus citations

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.

Original languageEnglish
Pages (from-to)79-103
Number of pages25
JournalMethods in Molecular Biology
Volume1498
DOIs
StatePublished - Jan 1 2017

Keywords

  • CRISPR/Cas9
  • Gene editing
  • Genetic transformation
  • Protozoan parasites
  • Selectable markers

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