A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe rate at which the genomes of intracellular malaria parasites can be modified has remained largely unchanged since methods for gene targeting by homologous recombination were developed in Plasmodium (). Some notable advances have recently improved transfection efficiency in P. falciparum through the application of zinc finger nucleases () and CRISPR-Cas9 (). However, no currently available method is efficient enough to enable reverse genetic screens, and transposon mutagenesis in P. falciparum is at present well short of genome saturation (). As a result, more than half of the protein coding genes in Plasmodium genomes still lack functional annotation.Genome-wide collections of mutants or genetic modification vectors have greatly facilitated the discovery of gene functions in model organisms (). In malaria parasites, in contrast, efforts to scale up reverse genetics have suffered from a combination of low rates of homologous recombination and a high content of adenine and thymine (A+T) nucleotides that renders Plasmodium DNA difficult to engineer in E. coli. A malaria parasite of rodents, P. berghei, offers the most robust system for genetic manipulation with relatively high transfection efficiency (). In this species homologous integration can be boosted further by transfecting linear vectors with long (4–8 kb) homology arms (). Despite its high A+T content (>77%), P. berghei genomic DNA (gDNA) can be propagated efficiently in E. coli as large genomic inserts of up to 20 kb using a low-copy bacteriophage N15-derived linear plasmid with covalently closed hairpin telomeres (). In contrast to high-copy circular plasmids, an N15-based arrayed gDNA library achieved nearly complete genome coverage with sufficient insert size to represent the majority of P. berghei genes in their entirety. Clones from this library can be converted into gene targeting and tagging vectors in 96 parallel liquid cultures using robust protocols (), which exploit highly efficient homologous recombination mediated by the Red/ET recombinase system of lambda phage in E. coli ().To accelerate the functional analysis of all P. berghei genes we here present a genome-scale community resource of long-homology genetic modification vectors that are individually quality controlled by sequencing and carry gene-specific molecular barcodes. The availability of more than 2,000 genome modification vectors raises the possibility of generating a large library of cloned and genotyped P. berghei mutants of the type that has enabled global genetic screens in yeast (). However, in P. berghei the lack of continuous in vitro culture of blood stages would limit the utility of such a clone collection. Signature-tagged mutagenesis, whereby thousands of mutants are simultaneously screened in a pooled approach (), therefore offers a more attractive strategy for scaling up reverse genetics in P. berghei.We have used the modification vector resource to enable such systematic screens for a Plasmodium parasite. We demonstrate that cotransfecting multiple gene knockout vectors in the same electroporation reproducibly generates complex pools of barcoded P. berghei mutants, and develop a barcode sequencing (barseq) approach () to phenotype the growth rates of all mutants within the pool over the course of an infection. To validate the approach, we compared a barseq knockout screen of protein kinases with the conventional kinome screen by . This comparison showed high reproducibility with previous data, but the sensitivity and robustness of the barseq approach also identified additional targetable genes. Our analysis demonstrates the power of barseq screening to robustly provide growth-rate phenotypes for dozens of mutants in single mice, and opens up the possibility for large-scale reverse genetic screens for multiple areas of Plasmodium biology.