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| Status |
Public on May 01, 2020 |
| Title |
Exploring the var gene interactome with CRISPR/dCas9 |
| Organism |
Plasmodium falciparum |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Expression profiling by high throughput sequencing
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| Summary |
Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.
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| Overall design |
An enzymatically "dead" Cas9 (dCas9) protein with a C-terminal 3xHA tag protein was guided by a sgRNA to either the promoter or intron of var genes. To confirm binding of dCas9 to the anticipated target sites, dCas9-ChIP sequencing was performed 14 hours post infection. As input control, DNA collected prior to dCas9 immunoprecipotation was sequenced. To identify RNAs associated with the target loci, the same dCas9-ChIP experiment was performed followed by RNA extraction and sequencing. For each sample used for dCas9-RIP, an anti-HA and anti-IgG (negative control) immunoprecipitation was performed.
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| Contributor(s) |
Bryant JM, Baumgarten S, Scherf A |
| Citation missing |
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| BioProject |
PRJNA529754 |
| Submission date |
Apr 05, 2019 |
| Last update date |
May 02, 2020 |
| Contact name |
Sebastian Baumgarten |
| E-mail(s) |
sebastian.baumgarten@pasteur.fr
|
| Organization name |
Institut Pasteur
|
| Department |
Parasites and Insect Vectors
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| Lab |
Biology of Host-Parasite Interactions
|
| Street address |
25, Rue du Dr. Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
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| Platforms (1) |
| GPL21298 |
Illumina NextSeq 500 (Plasmodium falciparum) |
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| Samples (5)
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| Relations |
| SRA |
SRP189826 |
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