State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
Nat Biotechnol. 2020 Dec;38(12):1460-1465. doi: 10.1038/s41587-020-0566-4. Epub 2020 Jun 29.
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.
短插入和缺失可以使用 CRISPR-Cas 编辑器在植物基因组中产生，但在特定靶位点可靠地产生更大的缺失已被证明难以实现。我们报告了一系列 APOBEC-Cas9 融合诱导缺失系统 (AFID) 的开发，该系统将 Cas9 与人类 APOBEC3A (A3A)、尿嘧啶 DNA-糖基化酶和无嘌呤或无嘧啶位点裂合酶结合在一起。在水稻和小麦中，AFID-3 从 5'-去氨化的 C 碱基到 Cas9 切割位点产生缺失。在水稻和小麦原生质体（30.2%）和再生植物（34.8%）中使用 AFID-3 产生的大约三分之一的缺失是可预测的。我们表明，eAFID-3 中，AFID-3 中的 A3A 被截短的 APOBEC3B (A3Bctd) 取代，从首选的 TC 基序到双链断裂产生更均匀的缺失。AFIDs 可用于研究调控区域和蛋白质结构域，以改良作物植物。
