Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: ARSENEL episomally. (A) Schematic of ARSENEL. Editing of eGFP His93 (CAC) to Tyr93 (TAC), labeled in red, restores eGFP fluorescence (the target cytosine is underlined, and the PAM site is labeled in blue). Constitutive mCherry expression provides an internal control for quantification of eGFP editing. The inset cartoon depicts editing by a CBE with Cas9n (tan), gRNA (gray), DNA (black), and deaminase (orange/green). (B, C) Efficiencies of A3Bctd and AIDΔC CBEs, respectively, using fluorescent microscopy (Cytation imaging) to quantify editing of the four ARSENEL constructs following cotransfection of all reaction components into 293T cells (data points are mean ± SD of biologically independent triplicate experiments). At the final time point, for A3Bctd CBE, all meanwise comparisons are significant (p < 0.0001 by two-way ANOVA) except AC versus GC. For AIDΔC CBE, only two meanwise comparisons are significant (AC vs. CC and GC vs. CC with p < 0.05 by two-way ANOVA). Inset immunoblots show CBE expression (anti-Cas9) and tubulin as a loading control (different lanes of the same representative immunoblot are shown in B and C and split only for purposes of presentation). ARSENEL, APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels; CBE, cytosine base editor; CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein; PAM, protospacer adjacent motif; SD, standard deviation.

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Labeling, Fluorescence, Expressing, Control, Microscopy, Imaging, Construct, Cotransfection, Western Blot, Standard Deviation

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: ARSENEL chromosomally. (A, C) Efficiencies of A3Bctd and AIDΔC CBEs, respectively, using fluorescent microscopy (Cytation imaging) to quantify editing of the four chromosomally integrated ARSENEL constructs following cotransfection of CBE and gRNA plasmids into 293T cells (data points are mean ± SD of biologically independent triplicate experiments; most error bars are smaller than the symbols). At the final time point, for A3Bctd CBE, all meanwise comparisons are significant (p < 0.0001 by two-way ANOVA) except AC versus GC. For AIDΔC CBE, only two meanwise comparisons are significant (GC vs. TC and GC vs. CC with p < 0.01 by two-way ANOVA). Inset immunoblots show CBE expression (anti-Cas9) and tubulin as a loading control (different lanes of the same representative immunoblot are shown in A and C and split only for purposes of presentation). (B, D) Editing rates from results in A and C after linear regression calculations (ns = not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001 by ordinary one-way ANOVA). (E) A3Bctd CBE and AIDΔC CBE editing rate comparisons after linear regression calculations compiled from B, D (ns = not significant; ****p < 0.0001 by two-way ANOVA).

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Microscopy, Imaging, Construct, Cotransfection, Western Blot, Expressing, Control

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: On-target and bystander editing events in chromosomal editing reactions with ARSENEL system. (A) Schematic of the gRNA-displaced region in the ARSENEL constructs. The on-target cytosine (C9) is highlighted in red and bystander cytosines are marked by asterisks. The CGG PAM site is underlined. (B) Stacking bar graph representations of A3Bctd CBE on-target and bystander editing events using each of the four different ARSENEL constructs from direct Sanger sequencing of PCR amplicons derived from genomic DNA. Unedited (WT) cytosines are shaded blue, C-to-A edits shaded green, C-to-G edits shaded gray, and C-to-T edits shaded red. Error bars represent the differences between the means of two biologically independent replicates. (C) Editing allele frequencies and linkage analysis for individually cloned and Sanger-sequenced PCR products from the gRNA-targeted region of eGFP from A3Bctd CBE editing reactions (percentages determined from n = 50 independent sequences). No indel events were detected in 50 independent clones. (D, E) Stacking bar graphs of on-target C9 editing by A3Bctd CBE and AIDΔC CBE, respectively, from direct Sanger sequencing of PCR amplicons derived from genomic DNA. Unedited cytosines are shaded blue, C-to-A edits shaded green, C-to-G edits shaded gray, and C-to-T edits shaded red. Error bars represent the differences between the means of two biologically independent replicates.

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Construct, Sequencing, Derivative Assay, Clone Assay

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: ARSENEL episomally. (A) Schematic of ARSENEL. Editing of eGFP His93 (CAC) to Tyr93 (TAC), labeled in red, restores eGFP fluorescence (the target cytosine is underlined, and the PAM site is labeled in blue). Constitutive mCherry expression provides an internal control for quantification of eGFP editing. The inset cartoon depicts editing by a CBE with Cas9n (tan), gRNA (gray), DNA (black), and deaminase (orange/green). (B, C) Efficiencies of A3Bctd and AIDΔC CBEs, respectively, using fluorescent microscopy (Cytation imaging) to quantify editing of the four ARSENEL constructs following cotransfection of all reaction components into 293T cells (data points are mean ± SD of biologically independent triplicate experiments). At the final time point, for A3Bctd CBE, all meanwise comparisons are significant (p < 0.0001 by two-way ANOVA) except AC versus GC. For AIDΔC CBE, only two meanwise comparisons are significant (AC vs. CC and GC vs. CC with p < 0.05 by two-way ANOVA). Inset immunoblots show CBE expression (anti-Cas9) and tubulin as a loading control (different lanes of the same representative immunoblot are shown in B and C and split only for purposes of presentation). ARSENEL, APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels; CBE, cytosine base editor; CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein; PAM, protospacer adjacent motif; SD, standard deviation.

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Labeling, Fluorescence, Expressing, Control, Microscopy, Imaging, Construct, Cotransfection, Western Blot, Standard Deviation

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: ARSENEL chromosomally. (A, C) Efficiencies of A3Bctd and AIDΔC CBEs, respectively, using fluorescent microscopy (Cytation imaging) to quantify editing of the four chromosomally integrated ARSENEL constructs following cotransfection of CBE and gRNA plasmids into 293T cells (data points are mean ± SD of biologically independent triplicate experiments; most error bars are smaller than the symbols). At the final time point, for A3Bctd CBE, all meanwise comparisons are significant (p < 0.0001 by two-way ANOVA) except AC versus GC. For AIDΔC CBE, only two meanwise comparisons are significant (GC vs. TC and GC vs. CC with p < 0.01 by two-way ANOVA). Inset immunoblots show CBE expression (anti-Cas9) and tubulin as a loading control (different lanes of the same representative immunoblot are shown in A and C and split only for purposes of presentation). (B, D) Editing rates from results in A and C after linear regression calculations (ns = not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001 by ordinary one-way ANOVA). (E) A3Bctd CBE and AIDΔC CBE editing rate comparisons after linear regression calculations compiled from B, D (ns = not significant; ****p < 0.0001 by two-way ANOVA).

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Microscopy, Imaging, Construct, Cotransfection, Western Blot, Expressing, Control

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: On-target and bystander editing events in chromosomal editing reactions with ARSENEL system. (A) Schematic of the gRNA-displaced region in the ARSENEL constructs. The on-target cytosine (C9) is highlighted in red and bystander cytosines are marked by asterisks. The CGG PAM site is underlined. (B) Stacking bar graph representations of A3Bctd CBE on-target and bystander editing events using each of the four different ARSENEL constructs from direct Sanger sequencing of PCR amplicons derived from genomic DNA. Unedited (WT) cytosines are shaded blue, C-to-A edits shaded green, C-to-G edits shaded gray, and C-to-T edits shaded red. Error bars represent the differences between the means of two biologically independent replicates. (C) Editing allele frequencies and linkage analysis for individually cloned and Sanger-sequenced PCR products from the gRNA-targeted region of eGFP from A3Bctd CBE editing reactions (percentages determined from n = 50 independent sequences). No indel events were detected in 50 independent clones. (D, E) Stacking bar graphs of on-target C9 editing by A3Bctd CBE and AIDΔC CBE, respectively, from direct Sanger sequencing of PCR amplicons derived from genomic DNA. Unedited cytosines are shaded blue, C-to-A edits shaded green, C-to-G edits shaded gray, and C-to-T edits shaded red. Error bars represent the differences between the means of two biologically independent replicates.

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Construct, Sequencing, Derivative Assay, Clone Assay

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: A3Bctd loop 7 dictates substrate specificity and editing efficiency. (A) Ribbon schematic of A3Bctd (pink) bound to ssDNA (green) with loop 7 residues highlighted in magenta (PDB: 5TD5). Three H-bonds govern the selection of TC substrates including one mediated by water (red). (B, C) Efficiencies of A3Bctd-L7G and A3Bctd-D314E CBEs, respectively, using fluorescent microscopy (Cytation imaging) to quantify editing of the four chromosomally integrated ARSENEL constructs following cotransfection of CBE and gRNA plasmids into 293T cells (data are mean ± SD of biologically independent duplicate experiments, each with two technical replicates). For the L7G construct, the 72-h editing time point is significantly different for CC versus TC (p < 0.05) and, for the D314E construct, the same time point is not significant for CC versus TC (p > 0.05 by two-way ANOVA). Different lanes of the same representative immunoblot from reactions 72 h post-transfection are shown in (B, C) and split only for purposes of presentation. (D) Bar graphs comparing the editing rates for A3Bctd, A3Bctd-L7G, and A3Bctd-D314E CBEs after linear regression calculations (data from Fig. 2A–C; ns = not significant; *p < 0.05; ****p < 0.0001 by two-way ANOVA).

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Selection, Microscopy, Imaging, Construct, Cotransfection, Western Blot, Transfection

Journal: The CRISPR Journal

Article Title: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels

doi: 10.1089/crispr.2023.0027

Figure Lengend Snippet: Demonstration of a virus-encoded inhibitor of A3Bctd CBE editing. (A) Efficiencies of A3Bctd CBE and A3Bctd-L7G in the absence and presence of BORF2 using flow cytometry to quantify editing of the chromosomally integrated TC reporter construct following CBE, gRNA, and BORF2 (or control) plasmid cotransfection into 293T cells (data are mean ± SD of biologically independent duplicate experiments, each with three technical replicates; ns = not significant; and ****p < 0.0001 by two-way ANOVA). The corresponding immunoblots from a representative experiment are shown below (the upper band in the anti-FLAG blot is BORF2-FLAG and the lower band is a cross-reacting cellular protein). (B) Efficiencies of A3Bctd CBE in the presence of the indicated BORF2 constructs using flow cytometry to quantify editing of the chromosomally integrated TC reporter construct following CBE, gRNA, and BORF2 (or control) plasmid cotransfection into 293T cells (data are mean ± SD of biologically independent triplicate experiments, each with two technical replicates; ns = not significant; *p < 0.05; ****p < 0.0001 by two-way ANOVA). The corresponding immunoblots from a representative experiment are shown below (the upper band in the anti-FLAG blot is BORF2-FLAG and the lower band is a cross-reacting cellular protein).

Article Snippet: CBE constructs Human A3A (No. 109425; Addgene), 17 human eA3A, 8 human full-length A3B (No. 198889; Addgene), 17 human A3Bctd (No. 109426; Addgene), 17 human AIDΔC CBE (No. 198890; Addgene), 36 and rat A1 (No. 73021; Addgene, deposited by D. Liu lab) 13 CBEs have been reported.

Techniques: Virus, Flow Cytometry, Construct, Control, Plasmid Preparation, Cotransfection, Western Blot