In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology.

Q2 Agricultural and Biological Sciences
生物设计研究(英文) Pub Date : 2022-08-01 eCollection Date: 2022-01-01 DOI:10.34133/2022/9823578
Zhiyuan Niu, Zhixia Luo, Pengyang Sun, Linwei Ning, Xinru Jin, Guanxu Chen, Changjiang Guo, Lingtong Zhi, Wei Chang, Wuling Zhu
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引用次数: 1

Abstract

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 in vitro. 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 in vitro.

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利用基因指定区域泛编辑技术构建体外纳米文库。
骆驼状单结构域抗体片段(纳米体)是近年来治疗性生物制药和临床诊断试剂领域的一股新兴力量。迄今为止,几乎所有可用的纳米体都是通过动物免疫获得的,这是限制纳米体大规模应用的瓶颈。在这项研究中,我们开发了三种基因指定区域泛编辑(GDP)技术,以在体外引入纳米体互补决定区(CDR)的多个突变。包括G-四链体片段在CDR中的整合,其诱导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CDR靶向传统gRNA引导的碱基编辑器,这有效地使CDR多样化。最重要的是,我们开发了自组装的gRNA,它是通过重新编程的tracrRNA劫持内源性mRNA作为crRNA产生的。使用自组装gRNA引导的碱基编辑器,我们可以实现CDR的迭代多样化。我们相信,最后一项GDP技术在无免疫纳米体库的构建中非常有前景,而这一新型纳米体发现平台的全面开发可以实现纳米体的体外合成进化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
3.90
自引率
0.00%
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审稿时长
12 weeks
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