Gene Disruption of Honey Bee Trypanosomatid Parasite, Lotmaria passim, by CRISPR/Cas9 System

In this study, we first generated L. passim clone expressing fluorescent marker and then attempted to use CRISPR/Cas9 for the genome editing. We will discuss how these approaches can be used to better understand honey bee-trypanosomatid parasite interactions. Materials and Methods Culture of L. passim Lotmaria passim strain SF (PRA-403) was obtained from the American Type Culture Collection (ATCC) and cultured in the modified FP-FB medium (Salathe et al., 2012) at 25°C without CO2. To monitor the growth rate of L. passim, the parasites were first inoculated at 5 × 105/mL and their number during the culture was measured by CASY® Cell Counter together with Analyzer System Model TT (OMNI Life Science). Electroporation of L. passim Followed by the Single Clone Isolation Actively growing L. passim (107/mL) was collected, washed twice, and resuspended in 0.4 mL of Cytomix buffer without EDTA (20 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 25 mM HEPES, and 5 mM MgCl2, pH 7.6) (Van Den Hoff et al., 1992; Ngo et al., 1998). 2 × 107 parasites were electroporated with 10 μg of pTrex-Neo-tdTomato (Canavaci et al., 2010) or pTrex-b-NLS-hSpCas9 (Peng et al., 2015) using a Gene Pulser X cell electroporator (Bio-Rad). For co-transfection of sgRNA expression vector and donor DNA, 10 μg of the plasmid DNA and 25 μg of the linearized donor DNA were electroporated. The parasites and DNA were mixed well and maintained on ice for 15 min in a cu...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research