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Development of a Simple and Quick Method to Assess Base Editing in Human Cells

Lv, Xiujuan, Qiu, Kairui, Tu, Tianxiang, He, Xiaoxue, Peng, Yuchen, Ye, Jinbin, Fu, Junhao, Deng, Ruzhi, Wang, Yuqin, Wu, Jinyu, Liu, Changbao, Zhao, Junzhao, Gu, Feng·2020·Molecular Therapy Nucleic Acids
DOI 10.1016/j.omtn.2020.03.004PMC PMC7184106
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Figure 1 Schematic of NOTEPAD System and Reporter Cell Line (A) Schematic of the NOTEPAD system. The MCS sequence contains 20 restriction sites, and we designed 5 target sites. Site 1 and site 5 were on the (–) strands (purple base is PAM). (B) Schematic of the transfection experiment. We transfected the plasmids to HEK293 cells (250 ng templates, 250 ng BE expression plasmids, and 125 ng sgRNAs expression plasmids) or HEK293-ME cells (250 ng BE expression plasmids, and 125 ng sgRNAs expression plasmids). The percentage of EGFP (or EGFP disruption) was analyzed by flow cytometry (FCM) or the genomic DNA was isolated for further analysis. (C) Schematic of the HEK293-ME cell line generation. A lentivirus containing EFIα-MCS-EGFP-Puro cassettes was packaged for infecting HEK293 cells. After puromycin selection, colonies of HEK293-ME were picked under a fluorescence microscope.
Figure 1 Schematic of NOTEPAD System and Reporter Cell Line (A) Schematic of the NOTEPAD system. The MCS sequence contains 20 restriction sites, and we designed 5 target sites. Site 1 and site 5 were on the (–) strands (purple base is PAM). (B) Schematic of the transfection experiment. We transfected the plasmids to HEK293 cells (250 ng templates, 250 ng BE expression plasmids, and 125 ng sgRNAs expression plasmids) or HEK293-ME cells (250 ng BE expression plasmids, and 125 ng sgRNAs expression plasmids). The percentage of EGFP (or EGFP disruption) was analyzed by flow cytometry (FCM) or the genomic DNA was isolated for further analysis. (C) Schematic of the HEK293-ME cell line generation. A lentivirus containing EFIα-MCS-EGFP-Puro cassettes was packaged for infecting HEK293 cells. After puromycin selection, colonies of HEK293-ME were picked under a fluorescence microscope.CC BY-NC-ND · PubMed Central
Figure 2 The Editing Events of BE3 Detected with NOTEPAD System (A) The plasmids expressing PME templates, BE3, and sgRNAs were transfected into HEK293 cells. Genomic DNA was harvested and PCR was performed with primers (F1 and R1) to amplify the DNA sequence flanking of MCS. PCR products were treated with restriction enzymes and then separated with electrophoresis. The resistant-cleavage band sequence information was obtained via Sanger sequencing. (B) The plasmids expressing BE3 and sgRNAs were transfected into HEK293-ME cells. The same protocol was adopted as (A). Blue sequence is target site, purple base is PAM, and red base and ellipsis represent edited events.
Figure 2 The Editing Events of BE3 Detected with NOTEPAD System (A) The plasmids expressing PME templates, BE3, and sgRNAs were transfected into HEK293 cells. Genomic DNA was harvested and PCR was performed with primers (F1 and R1) to amplify the DNA sequence flanking of MCS. PCR products were treated with restriction enzymes and then separated with electrophoresis. The resistant-cleavage band sequence information was obtained via Sanger sequencing. (B) The plasmids expressing BE3 and sgRNAs were transfected into HEK293-ME cells. The same protocol was adopted as (A). Blue sequence is target site, purple base is PAM, and red base and ellipsis represent edited events.CC BY-NC-ND · PubMed Central
Figure 3 The Editing Results of ABEs Detected with NOTEPAD System (A) The plasmids expressing xCas9-ABE7.10 and sgRNAs were transfected into HEK293-ME cells, genomic DNA was harvested, and PCR was performed with primers (F1/R1) to amplify the DNA sequence flanking of MCS. PCR products were treated with restriction enzymes and then separated with electrophoresis. The resistant-cleavage band sequence information was obtained via Sanger sequencing. (B) The plasmids expressing ABE7.9 and sgRNAs were transfected into HEK293-ME cells. The same protocol was adopted as (A). Blue sequence is target site, purple base is PAM, and red base and ellipsis are edited events.
Figure 3 The Editing Results of ABEs Detected with NOTEPAD System (A) The plasmids expressing xCas9-ABE7.10 and sgRNAs were transfected into HEK293-ME cells, genomic DNA was harvested, and PCR was performed with primers (F1/R1) to amplify the DNA sequence flanking of MCS. PCR products were treated with restriction enzymes and then separated with electrophoresis. The resistant-cleavage band sequence information was obtained via Sanger sequencing. (B) The plasmids expressing ABE7.9 and sgRNAs were transfected into HEK293-ME cells. The same protocol was adopted as (A). Blue sequence is target site, purple base is PAM, and red base and ellipsis are edited events.CC BY-NC-ND · PubMed Central
Figure 4 The Editing Efficiency of BEs (A) The BEs mediated editing efficiency of sites on the plasmid (episomal). (B) The BEs mediated editing efficiency of sites in the genome.
Figure 4 The Editing Efficiency of BEs (A) The BEs mediated editing efficiency of sites on the plasmid (episomal). (B) The BEs mediated editing efficiency of sites in the genome.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Lv, X., Qiu, K., Tu, T., He, X., Peng, Y., Ye, J., Fu, J., Deng, R., Wang, Y., Wu, J., Liu, C., Zhao, J., & Gu, F.
Venue
Molecular Therapy Nucleic Acids
Publisher
Elsevier BV
Published
2020-06
Volume
20
Pages
580-588
ISSN
2162-2531
DOI
10.1016/j.omtn.2020.03.004
PMC
PMC7184106
Cited by
12
References
6
Language
en
Licence
Funders
  • National Natural Science Foundation of China (81201181)
  • Science and Technology Program of Zhejiang Province (2017C37176)

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CoChat. (2026). Citation check: Development of a Simple and Quick Method to Assess Base Editing in Human Cells. https://app.cochat.ai/papers/10.1016/j.omtn.2020.03.004
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Lv, X., Qiu, K., Tu, T., He, X., Peng, Y., Ye, J., Fu, J., Deng, R., Wang, Y., Wu, J., Liu, C., Zhao, J., & Gu, F. (2020). Development of a Simple and Quick Method to Assess Base Editing in Human Cells. Molecular Therapy Nucleic Acids. https://doi.org/10.1016/j.omtn.2020.03.004