Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope

Abstract

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.

Fig. 1. Chemical modifications of ABE mRNA and guide RNAs are critical for efficient base editing in cells.

a Diagrams of ABE RA6.3 mRNAs with sequence-optimization (uridine-depletion) and chemical modification (5-methoxyuridine). Red: “A”; Yellow: “U”; Green: “G”; Blue: “C”. b ABE mRNA with both 5-methoxyuridine modification and uridine-depletion showed the highest protein expression in HEK293T cells by western blot. The mass of transfected RA6.3 mRNA is as indicated. ABE plasmid and Cas9 mRNA served as controls. c Comparison of editing efficiency by plasmids of ABE and guide RNA (p_RA6.3 + p_sgRNA), ABE mRNA + tracrRNA/crRNA, and ABE mRNA + sgRNA in HEK293T cells. The target “A” sites are highlighted in red. PAM is underlined. Data represent mean ± SD. d, e, Comparison of editing efficiency by DNA and RNA-encoded ABE at two genomic sites in HEK293T cells. The target “A” sites are highlighted in red. PAM is underlined. c–e All mRNAs are 5moU-6.3. All guide RNAs are moderately modified RNA. Control group (Ctrl) is cells transfected with 500 ng GFP plasmid. Graphs show mean values. Data represent mean ± SD (n = 3 biologically independent samples). d P = 0.0079 (**), 0.0005(***), <0.0001 (****), <0.0001 (****); e P < 0.0001 (****) (two tailed t-test). Source data are provided as a Source Data file for b–e.

Fig. 2. RNA-encoded ABE restores CFTR function in human lung airway cells with W1282X mutation.

a Expression of GFP plasmid and mRNA in CFF-16HBEge W1282X cells. Twelve hours of post electroporation, bright field and fluorescence images were taken. Scale bar = 100 µm. b Protospacer sequence used to base edit W1282X. The pre-mature stop codon is highlighted in yellow. PAM sequence is underlined. Target “A9” is red and bystander “A5” is blue. c A-to-G conversion rate in W1282X cell pools electroporated with moderately modified sgRNA and either unmodified (unmodified-6.3) or modified (5moU-6.3) ABE mRNA. Data represent mean ± SD (n = 3 (unmodified-6.3), n = 4 (5moU-6.3) biologically independent experiments). *P = 0.0456. d CFTR protein expression in 5moU-6.3-treated W1282X cell pools by western blot. GAPDH is loading control. Experiments were done for three times, and one is shown. e CFTR protein expression in three single cell clones. f Genotypes of cell clones. Clone 1 has a bystander editing. Clone 3 has base editing only on the SV40 disrupted CFTR allele. g ABE restored CFTR-mediated Cl- transport in clones 1 and 2. Values of area under the curve (AUC) in h is shown. Data represent mean ± SD (n = 3 wells of cells examined in one experiment). N.S. not significant (two tailed t-test). h Representative traces from electrophysiology assays. Source data are provided as a Source Data file for c–e.

Fig. 3. Nanoparticle delivery of ABE mRNA to correct Fah splice-site mutation in vivo.

a Diagram of Fah splicing before and after correction by RA6.3. Protospacer sequence is shown below. Exon sequence is in upper case and intron sequence is in lower case. Target “A” is red and bystander “A” is blue. PAM sequence is underlined. b Tracking mice body weight ratio after removal of NTBC water. Body weight ratio is calculated over the body weight at day 0 of NTBC removal. c Immunohistochemistry staining and Hematoxylin and Eosin staining (H&E) of mouse liver sections. Mouse 1 and 2 denotes mice treated with LNP-5moU-6.3 and LNP-sgFah (end point 58 days). Untreated Fah mut/mut mouse was kept on NTBC water. Scale bar = 100 µm d RT-PCR results from treated mouse liver. PC (positive control) indicates samples from mouse treated with plasmids-delivered ABE and guide RNA through hydrodynamic injection. Three liver lobes (as 1, 2, and 3) per mouse were collected and analyzed. Wildtype Fah amplicon is 405 bp and mutant Fah (lacking exon 8) is 305 bp. e A-to-G conversion rate at Fah gene locus in mice livers. Three liver lobes per mouse were collected and analyzed. A6G/A9G indicates editing efficiency at bystander/target “A” sites. Data represent mean± SD (n = 6). Source data are provided as a Source Data file for b, d, e.