Niu Zhiyuan, Luo Zhixia, Sun Pengyang, Ning Linwei, Jin Xinru, Chen Guanxu, Guo Changjiang, Zhi Lingtong, Chang Wei, Zhu Wuling
Synthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, China.
Department of Oncology, Xinxiang First People's Hospital, The Affiliated People's Hospital of Xinxiang Medical University, Xinxiang 453000China.
Biodes Res. 2022 Aug 1;2022:9823578. doi: 10.34133/2022/9823578. eCollection 2022.
Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies . Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies .
骆驼科单域抗体片段（纳米抗体）近年来在治疗性生物制药和临床诊断试剂领域成为一股新兴力量。迄今为止，几乎所有可用的纳米抗体都是通过动物免疫获得的，这是限制纳米抗体大规模应用的一个瓶颈。在本研究中，我们开发了三种基因指定区域全编辑（GDP）技术，以在纳米抗体的互补决定区（CDR）中引入多个突变。包括在CDR中整合G-四链体片段，其可诱导CDR中的自发多个突变；然而，这些突变序列高度相似，导致CDR中缺乏序列多样性。我们还使用了靶向CDR的传统gRNA引导的碱基编辑器，其有效地使CDR多样化。最重要的是，我们开发了自组装gRNA，它是由重新编程的tracrRNA劫持内源性mRNA作为crRNA产生的。使用由自组装gRNA引导的碱基编辑器，我们可以实现CDR的迭代多样化。并且我们相信最后一种GDP技术在免免疫纳米抗体文库构建中极具前景，而这个新型纳米抗体发现平台的全面发展能够实现纳米抗体的合成进化。
