High-purity production and precise editing of DNA base editing ribonucleoproteins

Abstract

Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM-targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.

Fig. 1. Purification of ABE/CBE proteins in human cell expression system.

(A) Schematic of base editor purification (see Materials and Methods). ORF, open reading frame. (B) Purified base editors. The peak fraction of ABEmax or AncBE4max from size exclusion chromatography (SEC) was collected, concentrated, and analyzed by SDS–polyacrylamide gel electrophoresis (PAGE). AU, absorbance unit. (C and D) Comparison of base editing frequencies after RNP- and plasmid-mediated ABE delivery (C) or CBE delivery (D). Dots represent the mean value of three independent biological replicates for each target. Error bars represent the SEM of the dots. (E) Percentage of sequencing reads with specific edited nucleotides that have been converted from target Cs in HEK293T cells after RNP- and plasmid-mediated CBE delivery with or without UGI overexpression. Cobalt, C to T; green, C to G; red, C to A. Error bars represent the SEM of three independent biological replicates. (F) Viability of HEK293T cells after RNP- and plasmid-mediated ABE/CBE delivery and GFP vectors. Viability was determined using a CCK-8 assay. Bars represent mean values, and error bars represent the SEM of three independent biological replicates. **P < 0.01, comparing with the GFP (lab-made) by one-way analysis of variance (ANOVA) test with Dunnett’s multiple comparison. n.s., not significant.

Fig. 2. RNP- and plasmid-mediated editing characteristics and at human endogenous DNA target sites.

(A and B) Number of base conversions in ABE-edited alleles. Bars represent the ratio of the number of reads containing single (pink), double (yellow), or triple (blue) A conversions to the total number of reads containing A conversions. In (A), the five most common sequences (positions 1 to 20) at ABE_site 5 are visualized, with the frequency indicated to the right. Colored nucleotides represent edited sequences. (C and D) Number of base conversions in CBE-edited alleles at different target sites. Bars represent the ratio of the number of reads containing single (pink), double (yellow), or triple (blue) C conversions to the total number of reads containing C conversions. In (A) to (D), error bars represent the SEM of three independent biological replicates. (E) ABE editing efficiencies in HEK293T cells at different time points after transfection of the ABE-encoding plasmid. (F) Number of base conversions in ABE-edited alleles at different time points after transfection of the ABE-encoding plasmid. Error bars represent the SEM of two independent biological replicates. (G and H) Analysis of ABE abundance in HEK293T cells at different time points after transfection of the ABE protein (blue) or ABE-encoding plasmid (orange) in the absence of sgRNA. In (G), the points indicate the ABE abundance relative to the maximum abundance obtained for a given delivery method; in (H), the bars represent the ABE abundance relative to that obtained from plasmid-mediated expression at 6 hours after transfection. Dots (G) and bars (H) represent the mean value of two independent biological replicates.

Fig. 3. Delivery-dependent variations in ABE-mediated DNA off-target effects.

(A) Off-target DNA base editing in HEK293T cells after ABE RNP, ABE-encoding mRNA, and ABE-encoding plasmid delivery. A-to-G editing efficiencies (top) and the ratio of off-target over on-target A-to-G editing efficiencies (bottom) are shown. Bars represent mean values, and error bars represent the SEM of three independent biological replicates. (B) Overview of orthogonal R-loop assay. (C and D) sgRNA-independent off-target DNA editing frequencies detected by the orthogonal R-loop assay. Each R-loop was performed by cotransfection of ABE RNP or ABE-encoding plasmid in (C) and CBE RNP or CBE-encoding plasmid in (D) with HEK_site 2–targeting sgRNA and dSaCas9-encoding plasmid with SaCas9 sgRNA targeting R-loop 5 or 6. Bars represent mean values, and error bars represent the SEM of three independent biological replicates. (E) Overview of cytosine conversion by ABEs. (F) Editing efficiency of DNA C conversions in HEK293T cells after ABE RNP and ABE-encoding plasmid delivery was analyzed. Bars represent mean values, and error bars represent the SEM of three independent biological replicates.

Fig. 4. Delivery-dependent variations in ABE-mediated RNA off-target effects.

(A) Off-target RNA base editing in HEK293T cells after ABE RNP, ABE-encoding mRNA, and ABE-encoding plasmid delivery in the presence of sgRNA. Efficiencies of A-to-I mRNA editing are indicated. Delivery of a plasmid encoding GFP was used as a control. Bars represent mean values, and error bars represent the SEM of four independent biological replicates. (B) Off-target RNA base editing in HEK293T cells after ABE protein and ABE-encoding plasmid delivery in the absence of sgRNA. Efficiencies of A-to-I mRNA editing are indicated. Bars represent mean values, and error bars represent the SEM of four independent biological replicates.

Fig. 5. NG-ABEmax RNP–mediated correction of the disease-associated mutation in rd12 mice.

(A) Strategy for NG-ABEmax RNP–mediated correction of the nonsense mutation in Rpe65 in rd12 mice. (B) Efficiencies of NG-ABEmax RNP–mediated mutation corrections in rd12 mEFs using different sgRNAs. Control, NG-ABEmax without sgRNA. Bars represent the mean values of two independent replicates. gX19 and gX20, in vitro transcribed sgRNAs, with mismatched 5′ Gs, containing 19- and 20-nucleotide (nt) spacers, respectively; X19 and X20, chemically synthesized sgRNAs containing 19 and 20 nt spacers, respectively. (C) Schematic of NG-ABEmax RNP–mediated treatment of rd12 mice via subretinal injections. RNPs (yellow circles) were encapsulated into cationic lipid nanoparticles. (D) Representative confocal micrograph of RPE from rd12 mice at 6 hours after injection. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E) Efficiencies of NG-ABEmax RNP–mediated mutation corrections in rd12 mice (n = 8). Genomic DNAs from RPE from eyes injected or not with NG-ABEmax RNPs were analyzed to determine target A-to-G editing efficiencies. Bars represent mean values, and error bars represent the SEM of the eight independent replicates. ***P < 0.001 by Mann-Whitney test. (F) Relative expression of Rpe65 mRNA in RPE from rd12 mice injected with NG-ABEmax RNPs (n = 4). (G) Representative confocal micrographs of the RPE from rd12 mice at 5 weeks after injection. Scale bars, 10 μm. ***P < 0.001 by Kruskal-Wallis test with Dunn’s multiple comparison tests. Nontarget, rd12 mice injected with NG-ABEmax RNPs including nontargeting sgRNA; Target, rd12 mice injected with NG-ABEmax RNPs including target sgRNA.