1Synthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003 Henan, China
2Department of Oncology, Xinxiang First People’s Hospital, The Affiliated People’s Hospital of Xinxiang Medical University, Xinxiang 453000, China
| Received 23 Jan 2022 |
Accepted 09 Jun 2022 |
Published 02 Aug 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 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.
