The oligos for mutation and correctional sgRNA were synthesized, cloned into the pGL3-U6-sgRNA-PGK-puromycin (51133; Addgene) and pUC57-sgRNA expression vector (51132; Addgene), and in vitro transcribed as the reported protocol:17 (link) mutation sgRNA, 5′-CGCCAATGGTGTTAACACATAGG-3′; correctional sgRNA, 5′-CCGCCAATGGTGTTAACACgTAG-3′. Besides, the correctional sgRNA was also cloned into the pGL3-U6-sgRNA-PGK-GFP plasmid for the comparison of BE3, YE1-BE3, and YEE-BE3. The plasmids of Cas9 (44758; Addgene), BE3 (73021; Addgene), YE1-BE3 (85174; Addgene), and YEE-BE3 (85177; Addgene) were used, and these plasmids were transcribed in vitro as the reported protocol.17 (link) All of the transcribed RNA were stored at −80°C. The ssODN was synthesized at Sangon Biotech (http://www.sangon.com/) as the following sequence: 5′-GACGTATGGTGTTGGGTAAATCCGGGAGGACATTTGCATGTGAAGCCGCCAATGGTGTTAACACgTAGGAACTGGCAGTTGTGTTGCTTGGTTGCACACTCATCAAGATC-3′.
Zeng Y., Li J., Li G., Huang S., Yu W., Zhang Y., Chen D., Chen J., Liu J, & Huang X. (2018). Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos. Molecular Therapy, 26(11), 2631-2637.
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