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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Cell. Infect. Microbiol. | doi: 10.3389/fcimb.2019.00126

Gene disruption of honey bee trypanosomatid parasite, Lotmaria passim, by CRISPR/Cas9 system

  • 1Graduate School of Bioagricultural Sciences, Xi'an Jiaotong-Liverpool University, China
  • 2Xi'an Jiaotong-Liverpool University, China

Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an L. passim clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous miltefosine transporter and tyrosine aminotransferase genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.

Keywords: CRISPR/Cas9 applications, Honey bee, trypanosomatid, Lotmaria passim, Genome editing

Received: 23 Nov 2018; Accepted: 10 Apr 2019.

Edited by:

Martin C. Taylor, London School of Hygiene and Tropical Medicine (LSHTM), United Kingdom

Reviewed by:

Eva Gluenz, University of Oxford, United Kingdom
Shane Wilkinson, Queen Mary University of London, United Kingdom  

Copyright: © 2019 Kadowaki and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Tatsuhiko Kadowaki, Xi'an Jiaotong-Liverpool University, Graduate School of Bioagricultural Sciences, Suzhou, 464-8601, China, emi@agr.nagoya-u.ac.jp