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

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.


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All studies must disclose on these points even when the disclosure is negative. Cell line source(s) Authentication 2x10e5 cells were used for editing in culture system. We first performed pilot titration experiments to determine the sample size. And according to previous studies (Song et al, Nature Biomedical Engineering, 2019), we confirmed this sample size to be sufficient to ensure reproducibility. All cell samples were evaluated in at least biological triplicates (n = 3) to ensure the reproductability. For animal experiment, we described the size in the specific figure legend. The size is determined based on the availability of the mice and previous reports (Song et al, Nature Biomedical Engineering, 2019).
No data was excluded.
Experiments were done in biological triplicate in culture cells, n=3 ,on different days (every three days). All attempts at replication were successful, and standard deviations were in the expected ranges.
For all the culture-related experiments, after seeding cell into 12-well plate, we randomly decided which cells are for experiment group or control group. For mouse experiment, we randomly decide the mice treated for control or LNP.
It is not applied to molecular and cell experiments. All mouse work are blind.
Mouse anti-Cas9 antibody (A-9000-050); Mouse anti-Fumarylacetoacetate hydrolase antibody (ab83770); mouse anti-GAPDH (EMD, MAB347); mouse anti-CFTR (UNC-596) Mouse anti-Cas9 validated by manufacturer by western blotting against over-expressed spCas9 from HEK293T cell extract. Mouse anti-Fumarylacetoacetate hydrolase antibody valisted by manufacturer by immunohistochemistry with Human liver and mouse KO tissue Mouse anti-GAPDH: validated by manufacturer by western blotting from rat brain tissue lysates. mouse anti-CFTR: validated by manufacturer by western blotting against CFTR protein.