1. Volume 25, Issue 4, Pages 884-892.e3 (October 2018)
Intrinsic Nucleotide Preference of Diversifying Base Editors Guides Antibody Ex Vivo Affinity Maturation 
Liu Daisy Liu, Min Huang, Pengfei Dai, Tingting Liu, Shuangshuang Fan, Xueqian Cheng, Yaofeng Zhao, Leng-Siew Yeap, Fei-Long Meng 
Cell Reports 
Volume 25, Issue 4, Pages e3 (October 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 884-892.e3DOI: (10.1016/j.celrep.2018.09.090)
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3. Figure 1 Comparison and Development of Base Editing Tools
(A) Comparison of base editing tools in the same genomic context. Base editing tools are schematically illustrated at left. Mutation profiles (i.e., mutation frequency at each nucleotide along a DNA sequence) of a 100-bp GFP gene fragment generated by using different Base Editing tools with the same sgRNA are shown in middle. Mutate frequency are plotted as the mean percentage of sequences that contain mutation at the indicated nucleotide from three independent experiments. Green bars indicate SEM. Please note the difference of y axis. The sgRNA targeting to the bottom DNA strand is boxed with dashed lines. Positions of AGCT and WRC (W: A/T; R: A/G) motifs are marked with orange and yellow bars, respectively. Proportions of sequences, that harbor indicated number of mutations per read, are plotted as pie chart at right.
(B) Base editing tools developed or optimized in this study. Panels are illustrated as in (A).
(C) Mutation profiles of a 700-bp GFP gene generated by using DBE with one sgRNA (upper) or sgRNA pool (lower) are shown. The sgRNA position is indicated with green (targeting top strand) or red (targeting bottom strand) bar.
See also Figure S1.
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4. Figure 2 Intrinsic Nucleotide Preference of Diversifying Base Editors
(A) Mutation profiles of a 100-bp GFP gene fragment generated by using DBE-A3A, DBE-rAPOBEC1, DBE-A3BAct is shown. Positions of indicated motifs are marked in color. The sgRNA targeting the bottom DNA strand is boxed with dashed lines. Panels are illustrated as in Figure 1A.
(B) Intrinsic tri-nucleotide preference of recombinant rat APOBEC1, AID mutant proteins determined in vitro.
(C) Mutations profiles generated by using DBE-AIDmono and DBE-AID-3C/F as illustrated in Figure 1A.
(D) Mutation profiles generated by using DBE and nDBE with AIDmono (left) or A3A (right) in wild-type and UNG−/−MSH2−/−(UM−/−) HEK293T cells as illustrated in Figure 1A. See also Figure S2.
(E) Mutation spectrums generated by using different DBE system with catalytic-dead Cas9(DBE), Cas9 nickase(nDBE), AIDmono in WT, or UNG−/−MSH2−/−(UM−/−) HEK293T cells.
(F) Mutation frequencies at C’s inside of sgRNA protospacer sequences of different sgRNAs by using nDBE-AIDmono (left) or nDBE-A3A (right) are shown. Positions of preferred motifs are boxed with red lines and Cas9 nicking sites are marked with blue lines. Colored scale bars are shown on top. See also Figure S3.
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5. Figure 3 Antibody Gene Somatic Hypermutation in Cultured Cells
(A) Mutate profiles of VB1-8 passenger allele gene in germinal center B cells initiated with physiological AID (data retrieved from Yeap et al. [2015]) in HEK293T cells by using DBE-AIDmono with different pool of sgRNAs.
(B) Mutate profiles of VB1-8 passenger allele gene in HEK293T cells by using DBE-A3A, DBE-AID-3C, DBE-AID-3F.
(C) Mutate profiles of a mouse antibody IgH variable exon gene and its codon-optimized version in HEK293T cells by using DBE-AIDmono.
All panels are illustrated as Figure 1C.
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6. Figure 4 Antibody Affinity Maturation in Cultured Cells
(A) Schematic illustration of scFv expression construct. SP, antibody signal peptide; VH, antibody heavy chain variable gene; VL, antibody light chain variable gene; Linker, linker peptide; Cγ1, human IgG1 constant region gene; TM, transmembrane region; T2A, self-cleavage 2A peptide; Puro, puromycin resistant gene.
(B) Antibody affinity was measured by surface staining with NP and anti-IgG1 antibody. Fractions of cells expressing potential high (upper) or low (lower) affinity antibody are indicated. Three sequential transfections were performed for DBE-AIDmono or DBE-A3A, while “DBE-AID-A3A” cells was transfected with DBE-AIDmono twice and DBE-A3A once alternatively.
(C) Experiment procedure of multiple mutation-selection cycles to get enriched mutations.
(D) Enriched mutations in VH1-8 of NPhi cells at each mutation-selection cycle. Mutated nucleotides are labeled in red, and percentage of indicated base substitute among all mutations of the same nucleotide is shown in brackets. See also Figure S4.
(E) Experimental BLI setup and representative response curves fitted with a 1:1 binding model (red) to quantify binding affinity (KD) of wild-type and mutant B1-8 scFv. Concentrations of scFv represent 2-fold serial dilutions ranging from 100 nM to 6.25 nM.
(F) A strategy of antibody ex vivo affinity maturation with DBE. See Discussion for details.
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