Fig. 1: In vivo CRISPR screen of the Cryptosporidium pyrimidine salvage pathway.

a Schematic illustrates how a knockout vector is generated. The first Golden Gate reaction occurs between a Cas9 expression plasmid and a 300 bp unique segment (containing the two 50 bp homology arms (one of which serves dual function as the gRNA), a unique DNA barcode, BsmBI restriction enzyme sites and the tracrRNA). The second Golden Gate reaction, using the BsmBI restriction enzyme sites, inserts a variable selection/reporter cassette to generate a complete knockout vector. The knockout vector then contains all the machinery to disrupt a gene of interest by inserting a variable selection cassette and barcode at the genomic locus. b A knockout vector targeting thymidine kinase (cgd5_4440) was generated and transfected into C. parvum sporozoites that were used to infect Ifnγ^–/– mice under paromomycin selection. Faecal samples were collected and the luminescence in faecal material was monitored. Data shows the mean faecal luminescence from a pooled cage sample ( ± SEM of 2 technical replicates), n = 4 mice. c Alignment of reads from whole genome sequencing of the thymidine kinase knockout strain to the C. parvum IOWAII genome at the site of insertion. Note the complete lack of alignment to the PAM site, which is removed by the homologous repair event. d Overview of CRISPR screening method. Following construction of KO vectors (detailed in 1a), sporozoites are transfected with gene specific KO vectors and used to infect mice. Specific barcodes are then amplified via high fidelity PCR and used to calculate fold enrichment (log2[%barcode(output) / %barcodes(input)]) which measures the relative fitness contribution of each gene. e Mouse faecal material was collected, and luminescence was monitored in the pooled sample from the pyrimidine salvage pathway CRISPR screens at 1 and 2 KO vectors per gene. Data shows the mean faecal luminescence from a pooled cage sample ( ± SEM of 2 technical replicates), n = 4 mice. f Rank ordered fold enrichment scores from the 1 and 2 KO vectors per gene pyrimidine salvage CRISPR screens. The colour indicates the relative fitness contribution, with dark purple showing high fitness conferring and dark green showing low fitness conferring. g Comparison of the fold enrichment scores between the 1 and 2 KO vectors per gene pyrimidine salvage CRISPR screens. Confidence refers to the inverse of the 95% confidence interval when comparing the log2 fold change scores from each screen (see methods).