Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1–nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.
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Sequence Read Archive
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This research was supported by grants from the Institute for Basic Science (IBS-R021-D1) to J.-S.K. and ToolGen, Inc. (0409-20160107) to D.K. The plasmid encoding the His6-rAPOBEC1-XTEN-dCas9 protein (pET28b-BE1) was a gift from David Liu.
J.-S.K. is a founder of and shareholder in ToolGen.
Integrated supplementary information
Supplementary Figure 1 Comparison of BE3-associateded base editing efficiencies and Cas9-associated indel frequencies in human cells
(a) Base editing efficiencies obtained with BE1 (rAPOBEC1–dCas9), BE2 (rAPOBEC1–dCas9–UGI), and BE3 (rAPOBEC1–nCas9–UGI) at seven endogenous target sites in HEK293T cells. Base editing efficiencies were measured by targeted deep sequencing. Error bars indicate s.e.m. (b) Cas9 nuclease-driven mutation frequencies were measured by targeted deep sequencing at seven endogenous target sites in HEK293T cells. (c) A table showing target DNA sequences and mutation frequencies. The PAM is shown in blue. (d) A graph showing the rank order of indel frequencies or base editing efficiencies at seven endogenous target sites.
Specificities of BE3 and Cas9 examined using mismatched sgRNAs at the RNF2 site. Base editing efficiencies and indel frequencies obtained with mismatched sgRNAs were measured by targeted deep sequencing. The PAM is shown in blue. Red or black asterisks indicate mismatched sgRNAs that were highly active with BE3 but poorly active with Cas9 or vice versa, respectively. Error bars indicate s.e.m. (n = 3).
Supplementary Figure 3 Correlation between indel frequencies associated with Cas9 nucleases and base editing frequencies associated with BE3 using mismatched sgRNAs at the EMX1 (a), HBB (b), and RNF2 (c) sites.
The red dots indicate mismatched sgRNAs with which the relative frequency of BE3-associated base editing was more than three times higher than the relative frequency of Cas9 nuclease-associated indels and the blue dots indicate sgRNAs with which the relative frequency of Cas9 nuclease-associated indels was more than three times higher than the relative frequency of BE3-associated base editing.
Supplementary Figure 4 SDS-Polyacrylamide gel showing the integrity of Cas9 and BE3ΔUGI proteins before and after incubation with genomic DNA.
Supplementary Figure 5 Quantitative real-time PCR showing almost complete cleavage of genomic DNA at the on-target site by Cas9 or BE3ΔUGI plus USER.
Supplementary Figure 6 IGV images showing straight alignments of sequence reads at the 6 different on-target sites.
Supplementary Figure 8 Venn diagrams showing the number of sites with DNA cleavage scores over 2.5 identified by Digenome-seq of Cas9 nuclease- and BE3ΔUGI-treated genomic DNA.
Supplementary Figure 9 The number of total sites (red) and the number of PAM-containing sites with ten or fewer mismatches (blue) for a range of DNA cleavage scores.
Intact human genomic DNA (left) and genomic DNA digested by BE3ΔUGI and USER (right) were subjected to whole genome sequencing.
Supplementary Figure 10 Venn diagrams showing the number of PAM-containing homologous sites with DNA cleavage scores over 0.1 identified by Digenome-seq of Cas9- and BE3ΔUGI-treated genomic DNA.
Blue bars represent the number of homologous sites that differ from on-target sites by up to 6 nt. Red squares (BE3ΔUGI) and green triangles (Cas9) represent the fraction of Digenome-identified sites for a range of mismatch numbers.
Supplementary Figure 12 Significant correlation between the number of BE3ΔUGI- and Cas9-associated sites identified by Digenome 1.0 (a) and Digenome 2.0 (b).
Supplementary Figure 13 Significant correlation between the number of BE3ΔUGI-associated sites identified by Digenome 1.0 (a) or Digenome 2.0 (b) and the number of sites with 6 or fewer mismatches.
Supplementary Figure 14 Examples of Digenome-captured off-target sites associated only with Cas9, which contain no cytosines at positions 4-8.
Supplementary Figure 15 Base editing efficiencies at Digenome-captured sites associated only with 3 different Cas9 nucleases.
No substitutions were detectably induced by BE3 at these Cas9-associated sites. On-target sequences (EXM1_On, HBB_On, and RNF2_ON) are also shown.
Supplementary Figure 16 Base editing efficiencies of 6 different BE3 deaminases at Digenome-negative sites with ≤ 3 mismatches with respective on-target sequences.
No substitutions were detectably induced by BE3 at these sites. On-target sequences are also shown.
(a) IGV image showing straight alignments of sequence reads at the Dmd on-target site. (b) Three sites, including the on-target site, were identified by Digenome 2.0. (c) No off-target substitutions were detectably induced at the two candidate sites identified by Digenome-seq in NIH3T3 cells.
(a) Sequences of sgRNAs at the 5’ terminus. (b) Base editing efficiencies were measured at the EMX1 on- and off-target sites by targeted deep sequencing in HEK293T cells. The heatmap represents relative specificities of modified sgRNAs, compared to that of gX19 sgRNA. The specificity ratio was calculated by dividing (on-target frequency of modified sgRNA/off-target frequency of modified sgRNA) by (on-target frequency of gX19 sgRNA/off-target frequency of gX19 sgRNA).
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Kim, D., Lim, K., Kim, ST. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat Biotechnol 35, 475–480 (2017). https://doi.org/10.1038/nbt.3852
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