A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite

Ana Rita Gomes, Ellen Bushell, Frank Schwach, Gareth Girling, Burcu Anar, Michael A. Quail, Colin Herd, Claudia Pfander, Katarzyna Modrzynska, Julian C. Rayner*, Oliver Billker

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

67 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)404-413
Number of pages10
JournalCell Host and Microbe
Volume17
Issue number3
DOIs
Publication statusPublished - 11 Mar 2015
Externally publishedYes

Bibliographical note

Funding Information:
We thank Tom Metcalf for technical assistance, Mandy Sanders for coordinating sequencing experiments, Thomas Dan Otto for guidance on analysing whole-genome sequence data, and the Sanger Institute sequencing teams. A plasmid encoding Emerald fluorescent proteins was a kind gift from J. Liu. A.R.G received a EVIMalaR studentship funded through the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242095. Work at the Sanger Institute was funded by grants from the Wellcome Trust (098051) and the Medical Research Council (G0501670).

Publisher Copyright:
© 2015 The Authors.

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