Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.

Abstract

Short insertions and deletions can be produced in plant genomes using CRISPR-Cas editors, but reliable production of larger deletions in specific target sites has proven difficult to achieve. We report the development of a series of APOBEC-Cas9 fusion-induced deletion systems (AFIDs) that combine Cas9 with human APOBEC3A (A3A), uracil DNA-glucosidase and apurinic or apyrimidinic site lyase. In rice and wheat, AFID-3 generated deletions from 5'-deaminated C bases to the Cas9-cleavage site. Approximately one-third of deletions produced using AFID-3 in rice and wheat protoplasts (30.2%) and regenerated plants (34.8%) were predictable. We show that eAFID-3, in which the A3A in AFID-3 is replaced with truncated APOBEC3B (A3Bctd), produced more uniform deletions from the preferred TC motif to the double-strand break. AFIDs could be applied to study regulatory regions and protein domains to improve crop plants.