Fig. 2

The second CRISPR/Cas9-mediated mutagenesis of the wild-type glmyb2 gene in JK1. a Schematics showing the strategy to delete the remaining wild-type glmyb2 gene in JK1. The plasmid used for mutagenesis of the JK1 strain contains the blasticidin-S resistance (bsr) cassette between the up- and downstream regions of the glmyb2 gene and RG1 gRNA expression cassette. This plasmid was transfected into JK1 by electroporation, and the transfectants were selected with three series of limiting dilutions with 75 μg/ml of blasticidin. b PCR analysis of the glmyb2 locus of wild-type (WB) and mutant JK2. c Western blot analysis of WB and mutant JK2 Giardia using anti-GlCWP1 antibodies (1:10,000). Both WB and mutant JK2 incubated in the encystation medium for 48 h were used to prepare cell extracts to determine the level of GlCWP1. The same membrane was treated in stripping buffer and then reacted with antibodies specific to GlPDI1 (1:10,000). d Coverage plot of the target gene region on Giardia chromosome 5 at positions 51,829 through 55,605. The blue bars indicate gene annotations from GeneDB. Read alignment views of the glmyb2 gene region of whole genome sequence and an average depth of fragment reads