Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
Level 5 - mechanism / opinion, no new human data
Bench research in transformed cell lines (no human clinical subjects)
PubMed 27096365 · doi:10.1038/nature17946
What was done
Researchers developed programmable base editors by fusing CRISPR/Cas9 to a cytidine deaminase enzyme to directly convert cytidine to uridine (enabling C-to-T or G-to-A substitutions) within an approximate five-nucleotide window without double-stranded DNA cleavage or donor templates. Second- and third-generation editors incorporated uracil glycosylase inhibitor and a Cas9 nickase targeting the unedited strand. The constructs were tested for point-mutation correction efficiency and indel formation across four transformed human and murine cell lines.
What was found
The engineered base editors mediated target base conversion within a ~5-nucleotide window. In the four tested cell lines, second- and third-generation editors yielded permanent target correction rates of ~15% to 75% of total cellular DNA, with indel formation typically restricted to <=1%.
Why it matters
This paper established base editing as a method for precise single-nucleotide correction, circumventing the double-stranded DNA breaks and donor DNA templates that typically generate high frequencies of random insertions and deletions.
Limits
The study was conducted entirely in vitro in four transformed human and murine cell lines. The abstract does not report in vivo delivery, genome-wide off-target effects, bystander editing frequencies within the target window, or durability in primary cells.
Cited by
- supports David Liu at Harvard developed CRISPR base editors that change nucleotides at targeted sites without generating double-stranded DNA breaks by using a deaminase domain.