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

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.

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

“…CRISPR/Cas9-sgRNA plasmids designed to target near the 3’ end of CaM genes were generated with Q5 DNA mutagenesis (S1C Fig), using guidelines described previously [31, 65] and co-transfected with their corresponding 6HA tag amplicons containing 42 bp homology regions matching the gene of interest (Fig 1A). Resistant clones were isolated, confirmed by PCR, and tested by immunofluorescence assay (IFA).…”

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

“…We recently engineered an RHΔ hxgprt Δ ku80 line of T. gondii to stably express TIR1 from Oryza sativa (RH TIR1-3FLAG) (Brown et al ., 2017; Long et al ., 2017a). In this background, we were able to use CRISPR/Cas9 genome editing (Shen et al ., 2014; Sidik et al ., 2014; Shen et al ., 2017) to tag essential T. gondii genes of interest with AID-3HA or mini-AID(mAID)-3HA, regulate their expression with auxin, and identify phenotypes associated with their loss (Brown et al ., 2017; Long et al ., 2017a and 2017b). …”

Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System

“…The CRISPR-Cas9 system provides a novel and promising tool for editing T. gondii genes (Shen et al, 2014) and the genome (Sidik et al, 2014; Wang et al, 2016a; Shen et al, 2017). Using CRISPR-Cas9 to target T. gondii 's GRA genes may ultimately offer a new approach to achieving functional cure to toxoplasmosis.…”