Project Summary / Abstract The objective of the proposed research is to create and validate new insect cell lines that can be used to produce glycoproteins (proteins with attached sugars to their surface) specifically for structural biology research. We will engineer insect cell lines so they produce sugar structures that can be removed under non-denaturing conditions, thereby retaining the three-dimensional structure of the proteins. As a result, better quality structural data can be obtained for these proteins. This includes for example membrane glycoproteins, glycoproteins involved in diseases such as cancer, and glycoprotein complexes. We will achieve our objective through three Specific Aims: 1) In Aim 1, we will generate custom CRISPR-Cas9 vectors and use them to disrupt the MGAT1 genes in our proprietary Sf-RVN and Tn-NVN cell lines. We will then obtain single cell clones without wild-type MGAT1 gene sequences. 2) Next, in Aim 2, we will confirm these clones produce proteins with the expected glycosylation pattern, and screen for productivity, growth, and viability. We will produce a non-cGMP cell bank for the best performers for distribution and deposit cells at the ATCC. 3) Finally, in Aim 3, we will use the new cell lines to produce two commercial protein drugs that require mannose-terminated glycans for efficacy. We will confirm the new cell lines can provide the increased mannose content that is associated with efficacy. The significance of the proposed research is that although insect cells are the most commonly used eukaryotic system used to produce proteins for structural research, they are not suitable for the production of glycoproteins for this purpose – even though about one half of all proteins are glycoproteins! Apart from the utility of the new cell lines for structural biology research, we believe they will have value for the production of glycoprotein therapeutics (“biologics”) that rely on mannose-rich glycans for efficacy. Such biologics include enzyme replacement therapeutics (targeting macrophages), as well as immunostimulants (targeting dendritic cells). The innovative aspects of the proposed research are: 1) We will use our proprietary insect cell lines that are free of persistent adventitious viruses. Thus, the new cell lines will have a superior biosafety profile for the production of proteins therapeutics. 2) We will use our newly developed CRISPR-Cas9 tools that are the first to enable targeted gene knockouts in the cell lines most commonly used for the baculovirus / insect cell platform.