In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology
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 n...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Association for the Advancement of Science (AAAS)
2022-01-01
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| Series: | BioDesign Research |
| Online Access: | http://dx.doi.org/10.34133/2022/9823578 |
| _version_ | 1797281093554012160 |
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| author | Zhiyuan Niu Zhixia Luo Pengyang Sun Linwei Ning Xinru Jin Guanxu Chen Changjiang Guo Lingtong Zhi Wei Chang Wuling Zhu |
| author_facet | Zhiyuan Niu Zhixia Luo Pengyang Sun Linwei Ning Xinru Jin Guanxu Chen Changjiang Guo Lingtong Zhi Wei Chang Wuling Zhu |
| author_sort | Zhiyuan Niu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-07T16:51:34Z |
| format | Article |
| id | doaj.art-e8393658e30e4a15ac94a11f7b9489f3 |
| institution | Directory Open Access Journal |
| issn | 2693-1257 |
| language | English |
| last_indexed | 2024-03-07T16:51:34Z |
| publishDate | 2022-01-01 |
| publisher | American Association for the Advancement of Science (AAAS) |
| record_format | Article |
| series | BioDesign Research |
| spelling | doaj.art-e8393658e30e4a15ac94a11f7b9489f32024-03-03T05:18:09ZengAmerican Association for the Advancement of Science (AAAS)BioDesign Research2693-12572022-01-01202210.34133/2022/9823578In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing TechnologyZhiyuan Niu0Zhixia Luo1Pengyang Sun2Linwei Ning3Xinru Jin4Guanxu Chen5Changjiang Guo6Lingtong Zhi7Wei Chang8Wuling Zhu9Synthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaDepartment of Oncology, Xinxiang First People’s Hospital, The Affiliated People’s Hospital of Xinxiang Medical University, Xinxiang 453000, ChinaSynthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, ChinaCamelid 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.http://dx.doi.org/10.34133/2022/9823578 |
| spellingShingle | Zhiyuan Niu Zhixia Luo Pengyang Sun Linwei Ning Xinru Jin Guanxu Chen Changjiang Guo Lingtong Zhi Wei Chang Wuling Zhu In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology BioDesign Research |
| title | In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology |
| title_full | In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology |
| title_fullStr | In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology |
| title_full_unstemmed | In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology |
| title_short | In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology |
| title_sort | in vitro nanobody library construction by using gene designated region pan editing technology |
| url | http://dx.doi.org/10.34133/2022/9823578 |
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