Successful gene disruption as determined by barcode PCR in a large pool of gene-deletion mutants.
Successful gene deletion indicates that the gene is not essential for asexual blood stage growth/multiplication.

P. berghei blood stages were transfected with a large pool of barcoded disruption (gene-deletion) vectors. These disruption vectors contained long modification arms to efficiently target the genes of interest. In addition the vectors contain gene-specific molecular barcodes. Co-transfecting multiple gene-deletion vectors in the same electroporation reproducibly generates complex pools of barcoded P. berghei mutants. Unsuccessful gene disruption/deletion or successful gene disruption/deletion is determined by the absence or presence of the barcode in the population (as determined by barcode-specific PCR analysis).

This report is based on simultaneous phenotyping of mutants by barcode sequencing and is part of a large-scale genetic screen. The genotype of this mutant was not confirmed.

The mutant was not isolated/cloned from the mixed population (large pool) of mutants.

Asexual blood stage phenotype:
Growth rate phenotypes were obtained by counting barcodes on a next generation sequencer daily between days 4 and 8 post transfection. It is in the nature of the screen that genotypes of individual mutants were not validated.

• Essential: growth rate not significantly different from 0.1.
• Dispensable: growth rate not significantly different from 1 (corresponding to wild type).
• Slow: growth rate significantly above 0.1 but below 1 (p < 0.05).
• Fast: growth rate significantly above 1 (p < 0.05).
   

In a follow-up study (Stanway RR, Bushell E et al., 2019) pools were generated of about 1360 blood stage-viable knock-out mutants and these were analyzed for their phenotypes throughout the entire parasite life cycle. Using barcode sequencing changes were measured in the relative abundance of knock-out mutants during transitions from blood stages (B1) to midgut oocysts (MG), midgut oocysts (MG) to salivary gland sporozoites (SG) and from salivary gland sporozoites (SG) to blood stages (B2) in mice following injection of sporozoites. The change in relative abundance in the 3 following life cycle transitions (B1-MG; MG-SG; SG-B2) is shown as ‘non-reduced’, ‘reduced’ and ‘no power’.
Blood samples were collected from each mouse infected with pools of knock-out mutants to establish the starting composition of mutants after drug (pyrimethamine) selection (sample B1). 120-150 female mosquitoes were then allowed to feed on each mouse, and midguts (MG) from >30 mosquitoes were dissected 15 days post infection, followed by salivary glands (SG) collection at day 22 post infection from at least 60 mosquitoes. Half of these SG were used to prepare a barseq library to establish the composition of the mutant pool in SG, the other half to collect sporozoites to infect another mouse. From this mouse a blood sample (B2) was taken 5 days after intravenous injection of sporozoites to establish the composition of the mutant pool in B2, allowing assessment of parasite development in the liver. Thus the change in abundance of knock-out mutants were determined in the 3 life cycle transitions B1-MG (blood stages to midgut oocysts/sporozoites), MG-SG (midgut oocysts/sporozoites to salivary gland sporozoites and SG-B2 (salivary gland sporozoites to blood stages).

Cross-fertilization between different knock-out mutants in the midgut of mosquitoes limits the power of the screen to reveal gene functions during the subsequent diploid and polyploid stages in the mosquito (i.e. zygotes, ookinetes and oocysts). For instance, knock-out mutants in which only one sex is sterile, can transmit their barcodes to the oocyst by inheritance through the fertile sex. As a result, reductions in barcode abundance for these sex-specific knock-out mutants often did not reach significance at the B1-MG conversion. Genes that are known to be essential for the diploid/polyploid ookinete/oocyst were also generally not recapitulated in the screen, presumably due to heterozygous rescue as a result of cross-fertilization. While these observations highlight the need for future screens to be designed specifically to reveal sexual and mosquito stage phenotypes, they also rationalize how knock-out alleles of genes functioning in fertility or ookinete/oocyst development can be transmitted to salivary gland sporozoites to reveal additional gene functions after sporozoite transmission to the vertebrate host.
Additionally the SG-B2 conversion rates were corrected using the known blood stage growth rate of each mutant to detect pre-erythrocytic/liver phenotypes more specifically.

The project website http://plasmogem.sanger.ac.uk/ has further information on methods and vector designs, provides tools for data visualisation and analysis, and allows researchers to request vectors to recreate mutants, confirm individual phenotypes and conduct more in-depth analyses.