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.

可以使用CRISPR–Cas编辑器在植物基因组中产生短插入和缺失，但是事实证明很难在特定目标位点可靠产生较大缺失。我们报告了一系列APOBEC–Cas9融合诱导的缺失系统（AFIDs）的发展，该系统将Cas9与人APOBEC3A（A3A），尿嘧啶DNA-葡萄糖苷酶和嘌呤或嘧啶定点裂解酶结合在一起。在水稻和小麦中，AFID-3从5'脱氨基的C碱基到Cas9切割位点产生了缺失。使用AFID-3在水稻和小麦原生质体（30.2％）和再生植物（34.8％）中产生的缺失大约是可预测的。我们显示eAFID-3，其中AFID-3中的A3A被截短的APOBEC3B（A3Bctd）取代，从首选TC母题到双链断裂均产生更均匀的缺失。