Targeted protein editing technique in living mammalian cells by peptide-fused PNGase

Various precise gene editing techniques at the DNA/RNA level,driven by clustered regularly interspaced short palindrome repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology,have gained significant prominence.Yet,research on targeted protein editing techniques remains limited.Only a few attempts have been made,including the use of specific proteases and de-O-glycosylating enzymes as editing enzymes.Here,we propose direct editing of Nglycosylated proteins using de-N-glycosylating enzymes to modify N-glycosylation and simultaneously alter the relevant asparagine residue to aspartate in living cells.Selective protein deglycosylation editors were developed by fusing high-affinity protein-targeting peptides with active peptide:N-glycanases(PNGases).Three crucial cell membrane proteins,programmed cell death protein-1(PD-1),programmed cell death-1 ligand 1(PD-L1),and severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)spike protein,were chosen to be tested as a proof of concept.N-linked glycans were removed,and the relevant sites were converted from Asn to Asp in living mammalian cells,destabilizing target proteins and accelerating their degradation.Further investigation focused on SARS-CoV-2 spike protein deglycosylation editing.The collaboration of LCB1-PNGase F(PNGF)effectively reduced syncytia formation,inhibited pseudovirus packaging,and significantly hindered virus entry into host cells,which provides insights for coronavirus disease 2019(COVID-19)treatment.This tool enables editing protein sequences post-de-N-glycosylation in living human cells,shedding light on protein N-glycosylation functions,and Asn to Asp editing in organisms.It also offers the potential for developing protein degradation technologies.