State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
Cell Rep. 2018 Oct 23;25(4):884-892.e3. doi: 10.1016/j.celrep.2018.09.090.
Base editors (BEs) are emerging tools used for precision correction or diversifying mutation. It provides a potential way to recreate somatic hypermutations (SHM) for generating high-affinity antibody, which is usually screened from antigen-challenged animal models or synthetic combinatorial libraries. By comparing somatic mutations in the same genomic context, we screened engineered deaminases and CRISPR-deaminase coupling approaches and updated diversifying base editors (DBEs) to generate SHM. The deaminase used in DBEs retains its intrinsic nucleotide preference and mutates cytidines at its preferred motifs. DBE with AID targets the same hotspots as physiological AID does in vivo, while DBE with other deaminases generates distinct mutation profiles from the same DNA substrate. Downstream DNA repair pathways further diversified the sequence, while Cas9-nickase restricted mutation spreading. Finally, application of DBE in an antibody display system achieved antibody affinity maturation ex vivo. Our findings provide insight of DBE working mechanism and an alternative antibody engineering approach.
碱基编辑器（BEs）是新兴的工具，用于精确校正或多样化突变。它为生成高亲和力抗体提供了一种潜在的方法，通常从抗原挑战的动物模型或合成组合文库中筛选。通过比较同一基因组背景下的体细胞突变，我们筛选了工程化脱氨酶和 CRISPR-脱氨酶偶联方法，并更新了多样化碱基编辑器（DBE）以产生 SHM。DBE 中使用的脱氨酶保留其内在的核苷酸偏好，并在其首选基序中突变胞嘧啶。具有 AID 靶标的 DBE 与生理 AID 在体内靶向相同的热点，而具有其他脱氨酶的 DBE 则从相同的 DNA 底物产生不同的突变谱。下游 DNA 修复途径进一步多样化了序列，而 Cas9-尼克酶限制了突变的扩散。最后，DBE 在抗体展示系统中的应用实现了体外抗体亲和力成熟。我们的研究结果提供了 DBE 工作机制的深入了解和一种替代的抗体工程方法。
