• Home
  • Search Results
  • Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite.

Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite.

Molecular systems biology (2020-08-21)
Jessica M Bryant, Sebastian Baumgarten, Florent Dingli, Damarys Loew, Ameya Sinha, Aurélie Claës, Peter R Preiser, Peter C Dedon, Artur Scherf

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

Product Number
Product Description

Iodoacetamide, BioUltra
Anti-acetyl-Histone H3 (Lys9) Antibody, serum, Upstate®
DL-Dithiothreitol, for molecular biology, ≥98% (HPLC), ≥99% (titration)
D-(+)-Glucosamine hydrochloride, ≥99%, BioReagent, suitable for cell culture
Gentamicin solution, 50 mg/mL in deionized water, liquid, 0.1 μm filtered, BioReagent, suitable for cell culture
Glycine, suitable for electrophoresis, ≥99%
Saponin from quillaja bark, Sapogenin content ≥10 %
SYBR® Green I nucleic acid gel stain, 10,000 × in DMSO
D-Sorbitol, for molecular biology, ≥98%
Supeltex M-2 Double-Tapered Ferrule, I.D. 0.25 mm, 0.10 mm to like I.D. column, pkg of 2 ea
Anti-RLN3 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution