A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite.
The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission.
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Ana Rita
AR
Gomes
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Ellen
E
Bushell
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Frank
F
Schwach
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Gareth
G
Girling
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Burcu
B
Anar
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Michael A
MA
Quail
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Colin
C
Herd
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Claudia
C
Pfander
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
Katarzyna
K
Modrzynska
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK. Electronic address: julian.rayner@sanger.ac.uk.
Julian C
JC
Rayner
Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK. Electronic address: oliver.billker@sanger.ac.uk.
Oliver
O
Billker
D000818:Animals; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008288:Malaria / Q000469:parasitology / Q000662:veterinary; D051379:Mice; D010962:Plasmodium berghei / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D059386:Reverse Genetics / Q000379:methods*
D011494:Protein Kinases