Engineering Plasmodium genomes is rendered difficult by relatively low rates of homologous recombination. A double-strand break in the target gene introduced by CRISPR-Cas9 increases homologous recombination (PMID: 24880488; PMID: 24987097), as does the use of longer homology arms. PlasmoGEM solved the problem of making long-arm vectors by overcoming a roadblock for engineering P. berghei DNA in E. coli at genome scale, despite the AT-rich and repetitive nature of the parasite’s genome (PMID: 22020067). Each PlasmoGEM vector carries a unique molecular barcode to count individual mutants in a mixture of transfectants. A large resource of efficient, barcoded vectors now enables genetic screens in P. berghei (PMID: 25732065).
CRISPR-Cas9 made a huge difference for knocking out individual genes, but since DNA repair in Plasmodium is largely homology driven, repair templates in the form of knock-out or knock-in vectors are still needed even with Cas9 mediated approaches. Barcoded PlasmoGEM vectors therefore remained the method of choice for simultaneous genetic screens in P. berghei.
The aim of PlasmoGEM was to create a community resource of arrayed and barcoded library of vectors with around 10 times longer-than-usual homology arms. The resulting increase in integration rate makes it possible to combine dozens of vectors in a single transfection to generate pools of mutants which can be phenotyped simultaneously by counting barcodes on a sequencer.
A major roadblock came from the fact that high-copy, circular plasmids in E. coli cannot maintain sufficiently large inserts of AT-rich and repetitive P. berghei gDNA to construct vectors with long (>3kb) homology arms robustly. A breakthrough came with a linear phage N15-based E. coli vector commercialized under the name pJAZZ (PMID: 20040575), with which suitable genomic libraries of P. berghei could finally be created, presumably because linear DNA does not exist in a supercoiled state, in which AT rich sequences are prone to break and recombine. However, even with linear phage vectors, not all Plasmodium genomic sequences were cloneable or could be engineered. The coverage of PlasmoGEM libraries therefore remains incomplete.
Once arrayed libraries of gDNA segments were available, recombinase-mediated engineering using the Red-ET system (PMID: 9771703) proved a robust method to turn gDNA sequences into genetic modification vectors on 96-well plates (Pfander et al., 2011). Recombineering reagents, protocols, and know-how were readily available at the Sanger Institute from the cloning pipeline supporting a large mouse knock-out programme (PMID: 21677750).
Many hundreds of PlasmoGEM vectors have now been shared with research labs worldwide to create individual mutants, and all available P. berghei vectors are used in barcode screens. Their long homology arms enable efficient genomic integration, and we rarely see extrachromosomal maintenance of unintegrated vectors, although strong and extended selection can select for integration into local duplications of essential genes that leave one copy of the target intact (PMID: 25662778).
With PlasmoGEM vectors, the composition and complexity of a pool of mutants can be adjusted to the biological question and to cope with life cycle bottlenecks. This is a major advantage over transposon-mediated, i.e., random, signature-tagged mutagenesis.
The production of gene disruption vectors with improved transfection efficacy has allowed for scaling up of phenotyping of P. berghei mutants using barcode sequencing technology (PMID: 25662778).
The simultaneous phenotyping of mutants in pools has advantages beyond the sheer breadth and unbiased nature of genetic screens. The fact that all phenotype measurements are stringently controlled within the same mouse or mosquito by dozens of other mutants in the same host leads to strikingly more precise phenotype measurements than comparing individual, clonal infections between different hosts, which suffer from a high degree of variance.
Blood stage phenotypic data for over 2500 P. berghei is now available pre-publication in the phenotypes section of our website.
All PlasmoGEM tools are free for use in non-commercial applications, but requestors will need to cover shipping costs. Please follow the instructions here to request resources.