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. 2021 Jan 8;11:625862. doi: 10.3389/fmicb.2020.625862

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Copyright © 2021 Zhao, Wang, Wang, Zhang, Jiang, Ding, Shen and Zhang.

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.

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Examination of multiplex genome editing. (A) Top left: K13 target sequence for generation of K13::Sir2B-mut^Cas9i highlighting the 20-nt guide sequence and the PAM (top). Modified locus shows the shield mutations (red) (bottom). Top right: DNA sequencing analysis of K13 shield mutations in K13::Sir2B-mut^Cas9i. Desired shield mutations are highlighted. Bottom left: DNA sequencing analysis of K13_C580Y mutation in K13::Sir2B-mut^Cas9i. C580Y mutation is highlighted. Bottom middle: DNA sequencing analysis of Sir2B shield mutations in K13::Sir2B-mut^Cas9i. Desired shield mutations are highlighted. The 20-bp target and shield mutations for Sir2B are the same as those used in single gene editing. Bottom right: Survival rates of RSA^{0– 3 h} for K13::Sir2B-mut^Cas9i clones and WT. Survival rates of mutant clones (mean 10.36, 9.62%) are significantly higher than WT (none), confirming the correlation between K13_C580Y mutation and artemisinin resistance. n = 2. WT, wild-type 3D7. (B) Top left: ARP4 target sequence for generation of ARP6-HA::ARP4-Ty1^Cas9i highlighting the 20-nt guide sequence and the PAM (top). Modified locus shows the shield mutations (red) (bottom). The 20-bp target and shield mutations for APP6 are the same as those used in single gene editing. Top right: Diagram illustrating the primers used for PCR verification of ARP6-HA::ARP4-Ty1^Cas9i. Primers for ARP6 (primers P8, P9, and P10) and ARP4 (primers P11, P12, and P13; in brackets) are indicated respectively. Bottom: PCR analysis of ARP6 (left) and ARP4 (right) in ARP6-HA::ARP4-Ty1^Cas9i and WT. The result confirms the presence of HA tag in ARP6 (primers P8/P9, 446 bp in the mutant; primers P8/P10, 740 bp in the WT and 800 bp in the mutant) and Ty1 tag in ARP4 (primers P11/P12, 502 bp in the mutant; primers P11/P13, 738 bp in the WT and 810 bp in the mutant). (C) DNA sequencing analysis of shield mutations in ARP6-HA::ARP4-Ty1^Cas9i. Desired shield mutations of ARP6 (left) and ARP4 (right) are highlighted. (D) Western blotting of ARP6-HA::ARP4-Ty1^Cas9i and WT. We used the mouse anti-HA antibody to check the ∼116 kDa ARP6-HA protein and mouse anti-Ty1 antibody to check the ∼60 kDa ARP4-Ty1 protein. The result shows the expression of ARP6-HA and Arp4-Ty1 proteins. Mutant, ARP6-HA::ARP4-Ty1^Cas9i. (E) IFA analysis of ARP6-HA::ARP4-Ty1^Cas9i. Nuclei were stained with Hoechst 33342 (blue fluorescence). The green fluorescence represents ARP4-Ty1 and the red fluorescence represents ARP6-HA. IFA reveals the co-localization of ARP4 and ARP6 in the nucleus. Bar = 5 μm.