Provided herein are enzymatic compositions for protein O-glycosylation and sialylation, methods and systems associated therewith. In particular, the composition for in vivo sialylation of therapeutic proteins. The composition comprises a polypeptide N-acetylgalactosaminyltransferase; a β-1,3-galactosyltransferase; an UDP-Glc/GlcNAc 4-epimerase; a disulfide bond isomerase; and an α-2,3-sialyltransferase or an α-2,6-sialyltransferase. Furthermore, provided herein are compositions for efficient and complete O-glycosylation and di-sialylation of therapeutic proteins.

The present invention aims to provide a saccharide composition suitable for a cyclic-tetrasaccharide-containing starch syrup which has low viscosity, low water activity, low coloration property, and low calorie content, and which is unlikely to cause precipitation of crystals of saccharides during storage, and to provide a use of the composition and a production method for the composition. The object is achieved by providing a cyclic-tetrasaccharide-containing saccharide composition having the following characteristics (1) to (3): (1) the saccharide composition includes a branched cyclic tetrasaccharide in addition to the cyclic tetrasaccharide, wherein the content of the cyclic tetrasaccharide with respect to the total solid content of the saccharide composition obtained by allowing glucoamylase and α-glucosidase to act on the above saccharide composition is 38% by mass or higher, on a dry solid basis; (2) the ratio of α-1,4-linked glucose in the total glucose residues constituting the saccharide composition in methylation analysis is over 9% and 15% or lower, and (3) the ratio of α-1,4,6-linked glucose in the total glucose residues constituting the saccharide composition in methylation analysis is less than 6%; and providing a use of the composition and a production method for the composition.

Provided is an SgRNA combination, comprising an SgRNA specifically targeting the GGTA1 gene, an SgRNA specifically targeting the CMAH gene and an SgRNA specifically targeting the β4GalNT2 gene. Also provided is a CRISPR/Cas9 vector combination, comprising a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector. Also provided is an applicaton of the CRISPR/Cas9 vector combination in knocking out the GGTA1 gene, the CMAH gene and the β4GalNT2 gene. The knockout rates of the three genes with the specifically targeted SgRNA sequences are respectively 56%, 63%, and 41%. A three genes knockoutpig can be obtained, wherein the three genes related to immune rejectionare knocked out, and heart valves of said pig can be acquired.

Provided is an SgRNA combination, comprising an SgRNA specifically targeting the GGTA1 gene, an SgRNA specifically targeting the CMAH gene and an SgRNA specifically targeting the 4GalNT2 gene. Also provided is a CRISPR/Cas9 vector combination, comprising a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a 4GalNT2-CRISPR/Cas9 vector. Also provided is an applicaton of the CRISPR/Cas9 vector combination in knocking out the GGTA1 gene, the CMAH gene and the 4GalNT2 gene. The knockout rates of the three genes with the specifically targeted SgRNA sequences are respectively 56%, 63%, and 41%. A three genes knockoutpig can be obtained, wherein the three genes related to immune rejectionare knocked out, and heart valves of said pig can be acquired.

Glycopeptides derived from short CD44 isoforms lacking amino acids encoded by exons 6-14; presenting one or multiple serine or threonine residues substituted with Tn (GalNAc-O-Ser/Thr) and/or sialyl-Tn (STn; Neu5Ac2-6GalNAc-O-Ser/Thr) antigens. Synthesizing the glycopeptides, including one-pot glycosylation of synthetic short isoform CD44 peptides through combination with nucleotide sugars and glycosyltransferases and purification of CD44s-Tn glycopeptides. Immunogenic chimeras derived from the CD44-Tn and/or STn glycopeptides, linked, in polyvalent form, to a carrier immunogenic protein, e.g., KLH CRM197. Conjugating the synthesized CD44s-Tn glycopeptides to the immunogenic protein carriers CRM197 and KLH, generating chimeric glycopeptides, termed CRM197-CD44s-Tn and KLH-CD44s-Tn. CD44-Tn/STn glycopeptides or compositions thereof for treating cancer and pre-neoplastic diseases, e.g., neoplastic diseases expressing short CD44 isoforms, through generating antibodies against cancer cells and treating/preventing cancer by vaccination. The glycopeptides, compositions, synthesis methods and uses can be employed in treating cancer, alone or in combination with immune checkpoint inhibitor therapy, chemotherapy, and radiotherapy.