Boosting genome editing of non-coding sequences in plants with glycosylase-mediated multi-nucleotide deletion editors

Compared with protein-coding sequences, non-coding sequences-such as promoters, untranslated regions (UTRs), microRNAs, and other regulatory ncRNAs-constitute the vast majority of the plant genome. They serve as critical regulators of gene expression and therefore represent promising targets for crop improvement. However, their study and manipulation remain challenging due to the lack of efficient tools for generating large genomic deletions. Here, we report that CRISPR-Cas9-based glycosylase base editors (gBEs) function primarily as highly efficient multi-nucleotide deletion editors (gMDEs) in plants, a role distinct from their predominant base-editing activity in mammals. This functional shift is likely driven by a preferential AP lyase repair pathway for glycosylase-generated abasic sites (apurinic/apyrimidinic or AP sites) in plant cells. Unlike its parental system, CRISPR-Cas9, gMDEs efficiently generate 6-20 bp deletions across protospacers in both rice and soybean. We demonstrate their versatility by generating a continuum of plant height variation through promoter editing of OsD18 and boosting grain size by disrupting regulatory elements in both the 5' and 3' UTRs of OsGLW7, which function through distinct regulatory mechanisms. This work establishes gMDEs as a versatile and precise genome editing platform for inducing multi-nucleotide deletions in plants, making them efficient tools for genetic perturbation, especially of non-coding sequences.