gRNA plasmids

By using the gRNA plasmid, the researchers successfully established a complete knockout line of the LmxBTN1 gene. However, the experimental results showed that even when the LmxBTN1 gene was completely knocked out, Leishmania was not significantly different from the wild-type strain in terms of in vitro growth and infectivity. This means that although the LmxBTN1 gene is upregulated at a specific pathogenic stage, it is not a gene required for parasite pathogenicity.(A Ishemgulova, et al.,2018)

Southern blot analysis of the BstE II digested L. mexicana genomic DNA of the WT, Cas9, and BTN1 ablated strains (labeled KO) with LmxM.22.0010 5’ UTR, LmxM.22.0010 3’ UTR and Puro probes.

A, Intensity of infection was assayed on days 2–3 and 7–8 p.i. and defined as weak (less than 100 promastigotes), moderate (100–1,000 promastigotes), or heavy (over 1,000 promastigotes), depending on the number of parasites per gut. Data are summarized from five independent biological replicates, numbers above each bar indicate the total number of dissected females. B, Quantitative PCR analysis of the L. mexicana load in the insect gut 7–8 days p.i. Boxplots are from five independent biological replicates and show 1st quartile, median, 3rd quartile, and 1.5× interquartile range values. C, Localization of parasites in sand fly gut 7–8 days p.i. (SV, stomodeal valve; TH,ABM, both thoracic and abdominal midgut; ABM, abdominal midgut). Numbers above each bar indicate the number of dissected females. D, Morphological analysis of Leishmania mexicana cells from thoracic midgut and stomodeal valve of infected sand fly females 7–8 days p.i. (LN, long nectomonade; SN, short nectomonade; ME, metacyclic promastigote).