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

作　　者：Zhiyuan Niu Zhixia Luo Pengyang Sun Linwei Ning Xinru Jin Guanxu Chen Changjiang Guo Lingtong Zhi Wei Chang Wuling Zhu 

机构地区：[1]Synthetic Biology Engineering Lab of Henan Province,School of Life Sciences and Technology,Xinxiang Medical University,Xinxiang,453003 Henan,China [2]Department of Oncology,Xinxiang First People’s Hospital,The Affiliated People’s Hospital of Xinxiang Medical University,Xinxiang 453000,China

出　　处：《BioDesign Research》2022年第1期208-216,共9页生物设计研究（英文）

基　　金：the National Natural Science Foundation of China(Grant 81902916);Natural Science Foundation of Henan Province(222300420513);National Key R&D Program of China(2019YFA0906000);National Natural Science Foundation of China(Grant 81903187 and 82003261).

摘　　要：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 designatedregion 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.

关 键 词：LIBRARY base BODY 

分 类 号：Q78[生物学—分子生物学]