CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells

Abstract

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications^1-4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities^5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.

Extended Data Fig. 1. On-target activities of nCas9 controls, ABE variants and more CBE variants tested for C-to-G editing in HEK293T cells

Bar plots showing the on-target DNA base editing frequencies induced by nCas9 negative controls, ABE and ABE variants and other CBE variants with seven gRNAs in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side. Arrowheads indicate cytosines showing C-to-G edits by CGBE1.

Extended Data Fig. 2. Indel frequencies of nCas9 controls, ABE variants and CBE variants tested for C-to-G editing in HEK293T cells

a,b, Dot plots representing percentage of alleles that contain an insertion or deletion across the entire protospacer from experiments with various base editor architectures reported in (a) Extended Data Fig. 1 or (b) Fig. 1b and Extended Data Fig. 1. Single dots represent individual replicates (n = 3 independent replicates).

Extended Data Fig. 3. On-target activities of nCas9 controls and CGBE1-related variants with 12 C6 gRNAs in HEK293T cells

Bar plots showing the on-target DNA base editing frequencies of nCas9 controls and CGBE1-related variants using 12 gRNAs for sites with a C at position 6 (C6-sites) in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side.

Extended Data Fig. 4. On-target activities of nCas9 controls and CGBE1-related variants with 6 non-C6 gRNAs in HEK293T cells and indel frequencies across 18 targeted sites

a, Bar plots showing the on-target DNA base editing frequencies of nCas9 controls and CGBE1-related variants using 6 gRNAs for sites with a C at position 4, 5, 7, or 8 (non-C6-sites) in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side. b, Dot plots representing percentage of alleles that contain an insertion or deletion across the entire protospacer from experiments with CGBE1-related variants reported in Fig. 2a and b and Extended Data Figs. 3 and 4a. Single dots represent individual replicates (n = 3 independent replicates).

Extended Data Fig. 5. Aggregated distribution of C-to-G editing frequencies across protospacer with CGBE1 and miniCGBE1 in HEK293T cells

a,b, Dot and box plots representing the aggregate distribution of C-to-G (yellow) editing frequencies per nucleotide across the entire protospacer from experiments performed with CGBE1 (a) and miniCGBE1 (b) with all 48 tested gRNAs. Boxes span the interquartile range (IQR; 25th to 75th percentile), horizontal lines indicate the median (50th percentile), and whiskers extend to ± 1.5 × IQR. Data points in plots represent full range of values plotted. Single dots represent individual replicates. The graphs were derived from the data shown in Fig. 3a,b (n = 4 independent replicates per site), and Supplementary Fig. 3a (n = 3 independent replicates per site).

Extended Data Fig. 6. On-target DNA editing activities of NG and VRQR variants of CGBE1 and miniCGBE1 in HEK293T cells

a, Bar plots showing the on-target DNA base editing frequencies induced by NG and VRQR variants of nCas9, CGBE1, and miniCGBE1 using 6 gRNAs that target AT-rich genomic loci with PAMs that are compatible with SpCas9-NG (NGT) and SpCas9-VRQR (NGAG) variants in HEK293T cells. Editing frequencies of four independent replicates (n = 4) at each base are displayed side-by-side. b, Dot plots representing percentage of alleles that contain an insertion or deletion across the entire protospacer from experiments with NG and VRQR variants of CGBE1 and miniCGBE1 reported in a. Single dots or triangles represent individual replicates (n = 4 independent replicates).

Extended Data Fig. 7. Comparing the editing activities of CGBEs and PEs in multiple human cell lines

a, Schematic of prime editing (PE) used to install a C-to-G substitution. PE fusion protein consists of an SpCas9-H840A nickase fused to an engineered Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The prime editing guide RNA (pegRNA) consists of a standard targetable SpCas9 gRNA that also harbors a 3’ extension containing a primer binding site (PBS) and a reverse transcription template (RTT) that encodes the desired edit. The PE2 system encompasses the prime editor fusion protein and a pegRNA. The PE3 system additionally includes a nicking gRNA (ngRNA). b, Bar plots showing the on-target DNA prime editing frequencies induced by nCas9(H840A), PE2 and PE3 using a pegRNA that targets FANCF site 1 across four human cancer cell lines. Gray overlay bars at top represent deletions at each nucleotide. Editing frequencies of four independent replicates (n = 4) for HEK293T cells or three independent replicates (n = 3) for K562, U2OS, and HeLa cells at each base are displayed side-by-side. Numbering on the bottom indicates the position of the base with 1 being the first nucleotide 3’ of the pegRNA/Cas9-induced nick. Arrowheads indicate guanines that exhibit desired G-to-T prime edits. c,d, Bar and dot plots representing the average on-target DNA prime editing and indel frequencies of PE2 and PE3 targeting FANCF site 1 for G-to-T prime editing (c; data from the same experiment as b) and HEK site 3 for PE-induced CTT insertion (d) in 4 cell lines. Single dots represent individual replicates (n = 4 for HEK293T and n = 3 for K562, U2OS, and HeLa cells). Error bars represent standard deviation (s.d.). Measure of center for the error bars = mean. e, Bar and dot plots showing the average on-target DNA C-to-G base or prime editing frequencies induced by CGBE1, miniCGBE1, PE2 or PE3 on four genomic target loci. Single dots represent individual replicates (n = 4 for HEK293T and n = 3 for K562, U2OS, and HeLa cells). A two-tailed Student’s t-test with p-values adjusted for multiple testing was used to calculate the shown p-values (p = 0.043 for both). Error bars represent (s.d.). Measure of center for the error bars = mean. f, Bar and dot plots representing the average frequency of alleles with indels (%) induced by pegRNAs and nicking gRNAs used in the experiments shown above (and FANCF site 1 +21 ngRNA control, Supplementary Table 9) with wild-type SpCas9 in HEK293T. pegRNAs/ngRNAs designed by Anzalone et al. (left) and by us (right) are separated by the dashed line. Single dots represent individual replicates (n = 3 independent replicates). Error bars represent (s.d.). Measure of center for the error bars = mean. ND, not done.

Figure 1:. Engineering of a C-to-G base editor

a, Schematic of potential cellular mechanisms and outcomes downstream of cytosine deamination by base editors. Uracil excision by endogenous uracil N-glycosylase (UNG, purple pentagon), nicking on the non-edited strand by Cas9 nickase, followed by DNA repair and replication can lead to diverse editing outcomes. C, Cytosine; G, Guanine; U, Uracil; T, Thymine; A, Adenine; UGI, uracil glycosylase inhibitor; AP lyase, apurinic/apyrimidinic site lyase; DSB, double strand break. b, Bar plots showing on-target DNA base editing frequencies with various base editor architectures using seven gRNAs targeting genomic sites in HEK293T cells. N and C in the base editor illustrations indicate amino-terminal and carboxy-terminal ends, respectively. Gray overlay bars at top represent deletions at each nucleotide. Target cytosines are highlighted. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side. Percentage values below specific cytosine bases indicate the average C-to-G editing observed (values below 3% not reported). Numbering on the bottom indicates position of the base in the protospacer with 1 being the most PAM-distal base. Arrowheads indicate cytosines with C-to-G edits.

Figure 2:. Additional characterization of CGBE1 on-target editing activities in HEK293T cells

a,b, Bar plots showing the on-target DNA base editing frequencies induced by BE4max(R33A) and CGBE1 using 12 gRNAs for sites with a C at position 6 (C6-sites; a) and 6 gRNAs for sites with a C at position 4, 5, 7, or 8 (non-C6-sites; b) in HEK293T cells. Editing frequencies of three independent replicates (n = 3) at each base are displayed side-by-side. c, Dot and box plots representing the combined distribution of C-to-G (yellow), C-to-T (red), C-to-A (green), and indel (gray) frequencies per nucleotide across the entire protospacer from experiments performed with BE4max(R33A) and CGBE1 using 25 guides. Boxes span the interquartile range (IQR; 25th to 75th percentile), horizontal lines indicate the median (50th percentile), and whiskers extend to ± 1.5 × IQR. Data points in plots represent full range of values plotted. Single dots represent individual replicates (n = 3 independent replicates per site). The graphs were derived from the data shown in Figs. 1b, 2a and b, and Extended Data Fig. 1.

Figure 3:. Comparison of CGBE1 and miniCGBE1 on-target editing activities in HEK293T cells

a,b, Bar plots showing the on-target DNA base editing frequencies of CGBE1 and miniCGBE1 using 19 gRNAs for sites with a C at position 6 (C6-sites; a) and 6 gRNAs for sites with a C at position 4, 5, 7, or 8 (non-C6-sites; b) in HEK293T cells. Editing frequencies of four independent replicates (n = 4) at each base are displayed side-by-side.