Journal: Nucleic Acids Research

Article Title: A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells

doi: 10.1093/nar/gky332

Figure Lengend Snippet: A real-time fluorescent reporter for APOBEC- and Cas9-mediated editing. ( A ) Schematic of the APOBEC- and Cas9-mediated editing (ACE) reporter in the context of a lentiviral construct with a CMV promoter that drives expression of a bicistronic message encoding mutant mCherry and wild-type eGFP. The sequence of the gRNA displaced DNA strand is shown below with flanking APOBEC 5′-TCA deamination hotspots (red), PAM sites, and 43 bp insertion labeled. See text for a description of editing, cleavage, and processing events required for reversion to mCherry-positive. Ribbon schematics of defective mCherry (gray) and functionally restored mCherry (red) with the flexible loop position of residue 59 shown (model based on pdb 2H5Q). ( B ) Schematic of an APOBEC–Cas9n/gRNA editosome engaging a DNA target. C-to-U editing occurs in the ssDNA loop displaced by gRNA annealing to target DNA. ( C ) Representative images of mCherry-positive 293T cells catalyzed by BE3 and mCherry codon 59-directed gRNA (#59-gRNA) but not with NS-gRNA (NS, non-specific; inset white bar = 30 μm). ( D ) Quantification of the base editing experiment in panel C ( n = 3; average ± SD).

Article Snippet: Key plasmids from our studies including the ACE reporter and the A3A and A3Bctd editing constructs are available through Addgene (ID 109425-109432).

Techniques: Construct, Expressing, Mutagenesis, Sequencing, Labeling, Residue

Journal: Nucleic Acids Research

Article Title: A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells

doi: 10.1093/nar/gky332

Figure Lengend Snippet: High-efficiency editing by human A3A and A3Bctd editosomes. ( A ) Representative fluorescence microscopy images of ACE-activated, mCherry-positive 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; inset white bar = 30 μm). ( B ) Quantification of the experiment in panel ‘A’ together with 2 independent parallel experiments ( n = 3; average ± SD). The corresponding immunoblots of expressed APOBEC–Cas9n-UGI constructs are shown below (low and high exposures to help visualize BE3) with HSP90 as a loading control. ( C ) Time course of ACE activation in 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; n = 3; mean ± SD; error bars smaller than symbols are not shown). ( D ) Titration data for 293T cells co-transfected with the ACE reporter, mCherry codon 59-directed gRNA, and different amounts of the indicated editosome constructs (100–600 ng; n = 3; mean ± SD). BE3i has an intron in the rat APOBEC1 portion of the construct, identical to the intron required for propagation of A3A and A3Bctd constructs in E. coli and for expression in mammalian cells.

Article Snippet: Key plasmids from our studies including the ACE reporter and the A3A and A3Bctd editing constructs are available through Addgene (ID 109425-109432).

Techniques: Fluorescence, Microscopy, Western Blot, Construct, Control, Activation Assay, Titration, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells

doi: 10.1093/nar/gky332

Figure Lengend Snippet: Chromosomal editing by A3A and A3Bctd editosomes. ( A ) Editing of single copy genomic ACE reporter by the indicated editosomes in 293T and HeLa cells ( n = 3, average ± SD). ( B ) Immunoblots corresponding to a representative experiment in panel A showing APOBEC–Cas9n-UGI expression levels and HSP90 as a loading control. ( C ) Sanger sequencing results for the gRNA-binding region of the ACE reporter recovered by high-fidelity PCR of mCherry-positive 293T. Mutated nucleotides are depicted in red and deleted nucleotides by hyphens. The number of times each sequence was recovered is indicated to the right.

Article Snippet: Key plasmids from our studies including the ACE reporter and the A3A and A3Bctd editing constructs are available through Addgene (ID 109425-109432).

Techniques: Western Blot, Expressing, Control, Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells

doi: 10.1093/nar/gky332

Figure Lengend Snippet: ACE reporter activation through Cas9 nucleolytic cleavage. ( A ) Quantification of ACE reporter activation in 293T cells 72 h after co-transfection of ACE reporter, mCherry codon 59 targeting gRNA or NS-gRNA, and A3A-Cas9n-UGI, A3Bctd-Cas9n-UGI, rat APOBEC1-Cas9n-UGI/BE3, Cas9, or Cas9n expression constructs ( n = 3; mean ± SD). ( B ) Anti-Cas9 and anti-HSP90 immunoblots from a representative experiment reported in panel A.

Article Snippet: Key plasmids from our studies including the ACE reporter and the A3A and A3Bctd editing constructs are available through Addgene (ID 109425-109432).

Techniques: Activation Assay, Cotransfection, Expressing, Construct, Western Blot

Journal: Nucleic Acids Research

Article Title: A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells

doi: 10.1093/nar/gky332

Figure Lengend Snippet: ACE enriches for base-editing events at heterologous genomic loci. ( A ) Schematic of a co-transfection experiment resulting in ACE reporter activation (yellow shading represents mCherry and eGFP double-positive cells). FANCF and the Pst I restriction assay used to quantify chromosomal base editing of this locus. Base editing events destroy the Pst I cleavage site and block cleavage of the 452 bp amplicon into 260 and 192 bp products. ( B ) Representative agarose gels images showing the results of FANCF base editing by A3A and A3Bctd editsomes in 293T cells. The percentage of base editing was calculated by dividing the percentage of substrate band by the total of substrate and product bands following Pst I cleavage for both unsorted and mCherry-positive cell populations. ( C ) Sanger sequencing results for the gRNA-binding region of the FANCF gene, which was recovered by high-fidelity PCR using genomic DNA from mCherry-positive 293T. Mutated nucleotides are highlighted in red and deleted nucleotides are indicated by hyphens. The number of times each sequence was recovered is indicated to the right.

Article Snippet: Key plasmids from our studies including the ACE reporter and the A3A and A3Bctd editing constructs are available through Addgene (ID 109425-109432).

Techniques: Cotransfection, Activation Assay, Restriction Assay, Blocking Assay, Amplification, Sequencing, Binding Assay

Journal: eLife

Article Title: Innervation modulates the functional connectivity between pancreatic endocrine cells

doi: 10.7554/eLife.64526

Figure Lengend Snippet: ( A ) Swimming activity of Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) zebrafish at 110 hpf was tracked pre- and post-blue light exposure for 5 min. Green lines show tracks at the end of the time-lapse. ( B, C ) Quantification of swimming activity of Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) zebrafish at 110 hpf; mean ± standard error of the mean (SEM), n = 21–23 animals, p values from two-way analysis of variance (ANOVA) with Holm–Sidak’s multiple comparisons test. ( D ) Schematic of optogenetic neural inhibition experiment.

Article Snippet: The Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) line was generated by Tol2 transgenesis of a 8.7 kb elavl3 promoter (Addgene: 59531, AgeI restriction enzyme digested) driving expression of sypb (Addgene: 74316), miniSOG2 (Addgene: 87410), P2A-mScarlet (gift from A. Beisaw) cloned with Cold Fusion (System Biosciences).

Techniques: Activity Assay, Inhibition

Journal: eLife

Article Title: Innervation modulates the functional connectivity between pancreatic endocrine cells

doi: 10.7554/eLife.64526

Figure Lengend Snippet: ( A ) Whole larva-free glucose-level measurements of Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) zebrafish at 110 hpf; mean ± standard error of the mean (SEM), n = 22–31 batches of 5 larvae per replicate, p values from two-way analysis of variance (ANOVA) with Holm–Sidak’s multiple comparisons test; see . ( B ) Normalized calcium traces of pancreatic islet cells (including delta, beta, alpha, and unidentified cells). Individual islet cells were assigned to a cell type and given a cell id; LG, basal condition; HG, glucose treated condition. ( C ) Correlation matrices of islet cell activity; LG, basal condition; HG, glucose treated condition; HG λ, post-blue light treatment. ( D ) Average beta cell correlation coefficients in individual larvae; n = 10 animals, p values from two-way ANOVA with Holm–Sidak’s multiple comparisons test. ( E ) Average homotypic (beta–beta) and heterotypic (alpha–beta) cell correlation coefficients with cell distance distribution from 1 (close) to 10 (far), mean and linear regression (solid lines) with 95% confidence intervals; n = 10 animals, p values of slope and intercept from simple linear regression. Fraction time analysis of heterotypic delta–beta, alpha–delta, and alpha–beta cell pairs for times when both are active ( F ), both are silent ( G ), and one is active and one is silent ( H ); mean ± SEM, n = 141–220 cell pairs, p values from two-way ANOVA with Holm–Sidak’s multiple comparisons test; magenta circle, active state; cyan circle, silent state. Figure 5—source data 1. . Whole larva-free glucose-level measurements of Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) zebrafish.

Article Snippet: The Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) line was generated by Tol2 transgenesis of a 8.7 kb elavl3 promoter (Addgene: 59531, AgeI restriction enzyme digested) driving expression of sypb (Addgene: 74316), miniSOG2 (Addgene: 87410), P2A-mScarlet (gift from A. Beisaw) cloned with Cold Fusion (System Biosciences).

Techniques: Activity Assay

Journal: eLife

Article Title: Innervation modulates the functional connectivity between pancreatic endocrine cells

doi: 10.7554/eLife.64526

Figure Lengend Snippet: List of zebrafish transgenic lines.

Article Snippet: The Tg(elavl3:sypb-miniSOG2-P2A-mScarlet) line was generated by Tol2 transgenesis of a 8.7 kb elavl3 promoter (Addgene: 59531, AgeI restriction enzyme digested) driving expression of sypb (Addgene: 74316), miniSOG2 (Addgene: 87410), P2A-mScarlet (gift from A. Beisaw) cloned with Cold Fusion (System Biosciences).

Techniques: Transgenic Assay, Marker