Journal: Nature biotechnology

Article Title: A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing

doi: 10.1038/s41587-020-0535-y

Figure Lengend Snippet: a , Schematic illustrating adenine base editor miniABEmax-V82G, cytosine base editor Target-AID, and dual-deaminase base editor SPACE. A-to-G (pink halo) and C-to-T (blue halo) base edits are illustrated. Light blue shape indicates Steptococcus pyogenes Cas9 (D10A) nickase, purple structure in Cas9 represents the guide RNA, pink circle represents the TadA 7.10 (V82G) adenosine deaminase monomer, blue triangle indicates the cytidine deaminase pmCDA1, and yellow circles represent uracil glycosylase inhibitors (UGIs). N and C labels denote the N and C termini of the base editor. b , Heat maps showing on-target DNA A-to-G (pink) and C-to-T (blue) editing frequencies of nCas9 (Control), miniABEmax-V82G, Target-AID, and SPACE with 28 gRNAs (n= 4 independent replicates). Editing windows shown represent the most highly edited adenines and cytosines, not the entirety of the protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal. c , Box and dot plots indicating the aggregate distribution of A-to-G (pink) and C-to-T (blue) edits across the entire protospacer with SPACE using 28 gRNAs. In box plots, the box spans the interquartile range (IQR) (first to third quartiles), the horizontal line shows the median (second quartile) and whiskers extend to ± 1.5 × IQR. Single dots represent individual replicates. Graph shows the same data as shown in b (n=4). d , Composition of alleles with frequencies of 1% or higher that result from SPACE-induced on-target DNA editing with a gRNA targeting HEK site 2 (ABE site 1). Data is shown for one replicate from the HEK site 2 on-target experiment shown in b . Numbering indicates the position in the protospacer with 1 being the most PAM-distal.

Article Snippet: The nCas9 (ABEmax without TadA domains; Addgene #123616) and GFP-only (enhanced GFP) constructs were used as negative controls.

Techniques: Control, Sequencing

Journal: Nature biotechnology

Article Title: A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing

doi: 10.1038/s41587-020-0535-y

Figure Lengend Snippet: a , Jitter plots show transcriptomic A-to-I (pink) and C-to-U (blue) mutations detected in RNA-seq experiments from HEK293T cells in which ABEmax, miniABEmax-V82G, Target-AID, or SPACE were co-expressed with either a HEK site 2 or RNF2 site 1 gRNA. GFP control cells express no gRNA. Data are shown from three independent replicates. n= number of combined adenines and cytosines modified. b , Heat maps showing A-to-G (pink) and C-to-T (blue) DNA off-target editing frequencies of nCas9 (Control), miniABEmax-V82G, Target-AID, or SPACE co-expressed in HEK293T cells with gRNAs targeted to HEK sites 2–4, EMX1 site 1, or FANCF site 1 (n= 4 independent replicates). The respective off-target editing windows for each site are shown. Location in the protospacer is indicated at the bottom with 1 being the most PAM-distal position. c , Circos plot showing amino acid changes that can be induced by SPACE (gray), including amino acid changes in specific codon context, that are uniquely enabled (blue). d , Bar plots showing computationally determined number of genes (y-axis) that could be targeted by SPACE to install 1–10 (or more) transcription factor (TF) binding sites (x-axis) in proximity of the transcription start site of coding genes in the human genome. Sites were filtered to contain a preferential SPACE editing window (C3-C4-A5 with 1 being the most PAM-distal position) and a canonical NGG PAM .

Article Snippet: The nCas9 (ABEmax without TadA domains; Addgene #123616) and GFP-only (enhanced GFP) constructs were used as negative controls.

Techniques: RNA Sequencing, Control, Modification, Binding Assay

Journal: Nature biotechnology

Article Title: A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing

doi: 10.1038/s41587-020-0535-y

Figure Lengend Snippet: a , Heat maps showing the on-target DNA A-to-G (pink) and C-to-T (blue) editing frequencies of nCas9 (Control), ABEmax, miniABEmax-V82G, Target-AID, or SPACE with HEK site 2 and RNF2 site 1 gRNAs (n=3 independent replicates) for the RNA-seq experiments shown in . Editing windows shown represent the most highly edited adenines and cytosines, not the entire protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal location. b , Histograms showing the total number of RNA A-to-I or C-to-U edits observed (y-axis) with different editing efficiencies (x-axis) for ABEmax, miniABEmax-V82G, Target-AID, or SPACE, each tested with the HEK site 2 and RNF2 site 1 gRNAs. n = number of modified adenines and cytosines. Experiments were performed in triplicate (data are from the same experiments shown in ). c , Heat maps showing on-target DNA A-to-G (pink) and C-to-T (blue) editing efficiencies of nCas9 (Control), miniABEmax-V82G, Target-AID, or SPACE with HEK sites 2–4, EMX1 site1, and FANCF site 1 gRNAs (n=4 independent replicates) for DNA off-target experiments shown in . Editing windows shown represent the most highly edited adenines and cytosines, not the entire protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal location. d , Circos plot showing 60 unique codon changes (with respect to the start codon) that can be induced by dual editing of adenines and cytosines by SPACE (grey), 18 of which (blue) lead to unique SPACE-inducible amino acid changes with respect to the original codon (also see and ).

Article Snippet: The nCas9 (ABEmax without TadA domains; Addgene #123616) and GFP-only (enhanced GFP) constructs were used as negative controls.

Techniques: Control, RNA Sequencing, Sequencing, Modification

Journal: Nature biotechnology

Article Title: A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing

doi: 10.1038/s41587-020-0535-y

Figure Lengend Snippet: Heat maps showing on-target DNA A-to-G (pink) and C-to-T (blue) editing frequencies induced by nCas9 (Control), co-expression of miniABEmax-V82G and Target-AID (ABE & CBE mix), or SPACE with 28 gRNAs (n=4 independent replicates). Editing windows shown represent the edited adenines and cytosines, not the entirety of the protospacer. Numbering at the bottom represents the position of the respective base in the protospacer sequence with 1 being the most PAM-distal location.

Article Snippet: The nCas9 (ABEmax without TadA domains; Addgene #123616) and GFP-only (enhanced GFP) constructs were used as negative controls.

Techniques: Control, Expressing, Sequencing