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.

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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.

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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. 

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