摘要：Camelid single-domain antibody fragments(nanobodies)are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent *** all nanobodies available to date have been obtained by animal immunization,a bottleneck restricting the large-scale application of *** 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 *** 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 *** also used CDR-targeting traditional gRNA-guided base-editors,which effectively diversify the *** most importantly,we developed the self-assembling gRNAs,which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as *** base-editors guided by self-assembling gRNAs,we can realize the iteratively diversify the *** 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.