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. 2021 Sep 29;9(2):e00738-21. doi: 10.1128/Spectrum.00738-21

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Copyright © 2021 Chiurillo et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 4 — Generation of analog-sensitive TcAEK1. (A) Schematic representation of CRISPR/Cas9-mediated strategy for TcAEK1 gatekeeper residue knock-in and C-terminal endogenous tagging. Cas9 introduced a DNA double-stranded break at nt +386 of the TcAEK1 ORF. Homologous-directed repair was induced cotransfecting epimastigotes with the DNA donor cassette, containing homologous regions to TcAEK1 (light blue) and to the TcAEK1 3′ UTR (light brown). The HDR determines integration of 3xc-Myc and antibiotic resistance gene (Pac, puromycin N-acetyltransferase) at the 3′ end of TcAEK1, as well as the mutation of the TcAEK1 gatekeeper residue codon sequence, which is included in the protospacer, as well as other silent mutations in the protospacer sequence avoiding being targets of the constitutively expressed Cas9. UTR, untranslated region; Igr, tubulin intergenic region. We used 3 different DNA donor cassettes to maintain the methionine in the wild-type TcAEK1 gatekeeper residue (M125) or to mutate it to the smaller alanine (M125A) or glycine (M125G) residues. M, M125; A, M125A; G, M125G. Horizontal green arrows indicate primers used for tagging verification. (B) PCR verification of cassette integration into TcAEK1 locus. TcAEK1 allele size: WT, 300 bp; 3xc-Myc-tagged, 1,581 bp. Lanes: 1, 1-kb plus ladder; 2, scrambled control; 3, TcAEK1^WT-3xc-Myc; 4, TcAEK1^M125A-3xc-Myc; 5, TcAEK1^M125G-3xc-Myc; 6, PCR negative control. (C) TcAEK1 endogenous tagging in gatekeeper residue mutants was verified by Western blotting using anti-c-Myc antibodies (c-My). Tubulin (Tub) was used as a loading control. Scr, scrambled, WT, TcAEK1^WT-3xc-Myc; Ala, TcAEK1^M125A-3xc-Myc; Gly, TcAEK1^M125G-3xc-Myc. (D) TcAEK1 gatekeeper in situ-mutated nucleotide sequence was confirmed in PCR-amplified wild-type and tagged size DNA fragments. Electropherograms show that, despite that only one allele was tagged at its C terminus, both alleles were mutated in each TcAEK1 gatekeeper mutant cell line: TcAEK1^M125A and TcAEK1^M125G. Asterisks (*) indicate mutations generated in the protospacer sequence. (E) Growth of control TcAEK1^WT (WT), TcAEK1^M125A (M125A), and TcAEK1^M125G (M125G) epimastigotes in LIT medium. One-way ANOVA with multiple comparisons was applied to growth rates calculated from each growth curve (n = 3, ***, P < 0.001).