A scalable pipeline for highly effective genetic modification of a malaria parasite.
In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
Claudia
C
Pfander
Burcu
B
Anar
Frank
F
Schwach
Thomas D
TD
Otto
Mathieu
M
Brochet
Katrin
K
Volkmann
Michael A
MA
Quail
Arnab
A
Pain
Barry
B
Rosen
William
W
Skarnes
Julian C
JC
Rayner
Oliver
O
Billker
D016054:DNA, Protozoan / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D018503:Genome, Protozoan / Q000235:genetics; D059765:Homologous Recombination; D008288:Malaria / Q000469:parasitology*; D010962:Plasmodium berghei / Q000235:genetics*
D016054:DNA, Protozoan; D004274:DNA, Recombinant