An arrayed CRISPR guide RNA library for Toxoplasma gondii
We have developed an arrayed CRISPR guide RNA library to rapidly generate fully customised CRISPR plasmid pools for knockout screening in Toxoplasma gondii. Our library currently consists of more than 12,000 single-stranded oligonucleotides, targeting ~2,400 genes with an average of five protospacer sequences per gene, and can be further expanded if needed. Selected oligonucleotides are sampled and pooled using a Echo Acoustic Liquid Handler in the Crick High Throughput Screening facility, then cloned in a pooled Gibson reaction into a CRISPR vector containing Cas9, the sgRNA constant sequence, and selectable markers for integration into the Toxoplasma genome.
The main advantage of our system is customisability: protospacer sequences can be precisely selected for targeted genes. In contrast to CRISPR libraries which are ordered as single pools or amplified by PCR from chips, these libraries can be fully tailored for an individual experiment.
This brings major advantages to our library in terms of scalability. In all CRISPR screening experiments, a major challenge is to maintain full complexity of genome-wide libraries with tens of thousands of guide sequences. By limiting the number of guides present in an experiment to only those that are required, we massively reduce technical challenge of transfection efficiency and passaging. Smaller target libraries additionally allow experiments which have a bottleneck in terms of cell number. Examples from our lab include in vivo screens, in which parasite number is highly limited by the lethal dose, and single-cell sequencing screens, in which a limited number of single cells can be captured.
Since only nanolitre volumes of each oligonucleotide are used, producing a CRISPR sub-library for a given experiment is highly cost-efficient. In addition, incorporating these sub-libraries into plasmid vectors ready for transfection is very rapid: we are able to go from a list of genes to target to a ready-to-transfect plasmid pool in less than one week.
One final advantage is adaptability: our system incorporates Cas9 into the same plasmid vector as the guide RNA, and so does not require Cas9-expressing strains. Our published vector can be used in any ΔHXGPRT Toxoplasma strain, but we are now also developing vectors that can be used in completely wild-type strains, including clinical and atypical isolates. We are also adapting our vector for use in Perturb-seq screens.
Our arrayed CRISPR library was developed by Joanna Young and Caia Dominicus, along with other members of the lab. For more details of how we have implemented it, please see the open access publication below. If you are interested in using our CRISPR library for your experiment, please get in touch with us to discuss possibilities
A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice.
Joanna Young, Caia Dominicus, Jeanette Wagener, Simon Butterworth, Xingda Ye, Gavin Kelly, Merav Ordan, Becky Saunders,Rachael Instrell, Michael Howell, Aengus Stewart, Moritz Treeck. Nature Communications volume 10, Article number: 3963 (2019).
Genetic tools for Toxoplasma gondii
A stable RH DiCre Δku80 cell line. While attempting to generate conditional knockout Toxoplasma cell lines for our studies using he well-established split Cre recombinase (DiCre) system, we observed a frequent loss of one of the DiCre subunits, resulting in poor excision of the targeted locus. To address this, Alex Hunt in our lab generated a new DiCre construct, DiCre-T2A, which expresses both DiCre subunits and the choloramphenicol acetyltransferase (CAT) selectable marker from a single promoter, separated by T2A skip peptides to produce the three separate proteins. This construct was inserted into the KU80 locus of the RH Δku80 ΔHXGPRT cell line. This second-generation DiCre line has excision efficiency of 98-99%, with less than 3% loss in efficiency after passage with extracellular stress for 65 days. In contrast, the original DiCre line lost 42% of excision capacity by day 65. For additional details, see the paper below. The parasite line is deposited at BEI resources (www.beirecouses.org) under the accession number NR-51627. We acknowledge that BEI does not ship to all countries. If you encounter issues receiving the line, contact us if you would like use to send you this line for your experiments.
Differential requirements for cyclase-associated protein (CAP) in actin-dependent processes of Toxoplasma gondii.
Alex Hunt, Matthew Robert Geoffrey Russell, Jeanette Wagener, Robyn Kent, Romain Carmeille, Christopher J Peddie, Lucy Collinson, Aoife Heaslip, Gary E Ward, Moritz Treeck. eLife 2019;8:e50598 (2019).
Genetic tools for Plasmodium falciparum
Conditional genome engineering using loxP sites in a small synthetic intron. Limitations of novel genetic tools in Plasmodium falciparum are mainly dictated by its low transfection efficiency. For that reason, facile application of the tightly controllable conditional Cre/lox system, has been hampered by the fact that flanking target genes with loxP sites can take many months. Matt Jones in our lab developed a strategy for easy and rapid conditional genome editing using a loxP-containing synthetic intron (loxPint), tailored for organisms like P. falciparum with short and few introns. We demonstrated correct splicing of these introns, and highly efficient excision of the surface protein MSP1, which has previously been refractory to genetic manipulation.
A marker-free P. falciparum cell line for conditional knockout across the life cycle. One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. Marta Tiburcio in our lab developed a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. This allows to test gene function of blood stage essential genes across the lifecycle. For further details, please see the paper below and contact us if you would like to us to send you this cell line.
Marta Tibúrcio, Annie S. P. Yang, Kazuhide Yahata, Pablo Suárez-Cortés, Hugo Belda, Sebastian Baumgarten, Marga van de Vegte-Bolmer, Geert-Jan van Gemert, Youri van Waardenburg, Elena A. Levashina, Robert W. Sauerwein, Moritz Treeck. mBio, 2019.