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Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

Akhila Bettadapur, Samuel S. Hunter, Charles G. Barbieri, Matthew L. Settles, Katherine S. Ralston
doi: https://doi.org/10.1101/2020.05.28.121780
Akhila Bettadapur
1Department of Microbiology and Molecular Genetics, University of California, Davis, USA
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Samuel S. Hunter
2Genome Center, University of California, Davis, USA
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Charles G. Barbieri
3SeqMatic, LLC, Fremont, California, USA
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Matthew L. Settles
2Genome Center, University of California, Davis, USA
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Katherine S. Ralston
1Department of Microbiology and Molecular Genetics, University of California, Davis, USA
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  • For correspondence: ksralston@ucdavis.edu
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Abstract

Entamoeba histolytica is a globally important pathogen that is dramatically understudied. Its challenging genome has limited tractability. To enable forward genetics, we constructed the first genome-wide E. histolytica RNAi knockdown mutant library. The library is designed to enable deep sequencing analysis for quantitative identification of mutants after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. We performed the first E. histolytica RNAi screen and identified slow growth mutants. Growth phenotypes were reproducible in independently generated mutants. Some of the genes targeted in slow growth mutants had annotated functions consistent with roles in growth or metabolism. Some targeted genes lacked annotation, supporting the power of forward genetics in uncovering gene function. This work opens up the possibility of applying genetics to improve understanding of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 29, 2020.
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Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth
Akhila Bettadapur, Samuel S. Hunter, Charles G. Barbieri, Matthew L. Settles, Katherine S. Ralston
bioRxiv 2020.05.28.121780; doi: https://doi.org/10.1101/2020.05.28.121780
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Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth
Akhila Bettadapur, Samuel S. Hunter, Charles G. Barbieri, Matthew L. Settles, Katherine S. Ralston
bioRxiv 2020.05.28.121780; doi: https://doi.org/10.1101/2020.05.28.121780

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