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

Cell Host & Microbe
Ana Rita GomesOliver Billker

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.

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Methods Mentioned

BETA
transfection
genetic modification
gene knockout
PCR
genotyping
transfections
transgenic
electrophoresis

Software Mentioned

Perl script
Plasmo GEM
Plasmo

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