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CRISPR-Cas9 Vectors for Genome Editing and Host Engineering in the Baculovirus–Insect Cell System

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CRISPR-Cas9 Vectors for Genome Editing and Host Engineering in the Baculovirus–Insect Cell System CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus–insect cell system Hideaki Mabashi-Asazumaa,1,2 and Donald L. Jarvisa,b aDepartment of Molecular Biology, University of Wyoming, Laramie, WY 82071; and bGlycoBac, LLC, Laramie, WY 82072 Edited by Vlad M. Panin, Texas A&M University, College Station, TX, and accepted by Editorial Board Member Peter Palese July 18, 2017 (received for review April 10, 2017) The baculovirus–insect cell system (BICS) has been widely used to insects naturally produce endogenously sialylated glycoproteins produce many different recombinant proteins for basic research (15–17). Nevertheless, this genetic capacity is not expressed in and is being used to produce several biologics approved for use in Sf9 or High Five cells. Insect cells also have a trimming enzyme, human or veterinary medicine. Early BICS were technically com- absent in mammalian cells, which antagonizes N-glycan elon- plex and constrained by the relatively primordial nature of insect gation (18–21). This enzyme, which is a specific, processing cell protein glycosylation pathways. Since then, recombination has β-N-acetylglucosaminidase called fused lobes (FDL), removes a been used to modify baculovirus vectors—which has simplified the terminal N-acetylglucosamine residue from trimmed N-glycan– system—and transform insect cells, which has enhanced its protein processing intermediates. This antagonizes elongation because glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific it eliminates the N-glycan intermediates used as substrates for genome editing are needed to facilitate further improvements in N-acetylglucosaminyltransferase II, which initiates the elonga- the BICS. Thus, in this study, we used various insect U6 promoters tion process. The inability of the BICS to produce mammalian- to construct CRISPR-Cas9 vectors and assessed their utility for site- type, elongated N-glycans is a major deficiency of this system specific genome editing in two insect cell lines commonly used as because these structures are required for clinical efficacy in hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit glycoprotein biologics (22). Because of its inability to synthesize an endogenous insect cell gene and alter protein glycosylation in these structures, it is widely believed that the BICS platform the BICS. could never be used for glycoprotein biologics manufacturing. As indicated above, this limitation has been addressed by using CRISPR-Cas9 | baculovirus | insect cells | genome editing | glycoengineering nonhomologous recombination to engineer insect cell N-glycosyla- tion pathways for mammalian-type N-glycan biosynthesis (reviewed he baculovirus–insect cell system (BICS), first described in in refs. 12–14 and 23). These efforts have yielded new, transgenic T1983 (1), has been used to produce thousands of different insect cell lines that can be used to produce recombinant glyco- recombinant proteins for diverse areas of biomedical research. proteins with fully elongated, mammalian-type N-glycans. However, Since 2009, the BICS has also been used to produce several bio- further glycoengineering is needed to create host cell lines that can logics approved for use in human or veterinary medicine (reviewed more efficiently process N-glycans in mammalian fashion and pro- in ref. 2). Thus, the BICS is an important recombinant protein duce homogeneously glycosylated proteins. These more refined production platform that has had and will continue to have a large glycoengineering efforts will require tools for site-specific genome and broad impact on basic research, biotechnology, and medicine. editing in the BICS, and fdl, which encodes an antagonistic func- Two precedents suggest the BICS would have even more impact if tion, will be a critically important target. it could be engineered to enhance its capabilities or extend its utility. The CRISPR-Cas9 system is a relatively new and exceptionally In the 1980s, the isolation of baculovirus expression vectors was a powerful tool for site-specific genome editing (24–26). CRISPR- highly inefficient, time-consuming, and frustrating process. However, Cas9 vectors have been constructed for and used in many different by the early 1990s, efforts to engineer the baculoviral genome in various ways had greatly simplified this process (3–5). These re- Significance finements effectively converted a complex system created in highly specialized laboratories to a routine tool that could be easily used in CRISPR-Cas9 is a powerful site-specific genome-editing tool many different laboratories. This was followed by efforts to enhance that has been used to genetically engineer many different the BICS by engineering host protein N-glycosylation pathways systems. However, this tool has not yet been adopted for (reviewed in ref. 6). However, host glycoengineering and other host use in the baculovirus–insect cell system, which is an im- improvement efforts have been limited to the use of nonhomologous portant recombinant protein production platform. Thus, we recombination to knockin heterologous genes at random sites created new CRISPR-Cas9 vectors that can be used for ge- (reviewed in ref. 7). This is because there have been no tools for site- nome editing in two relevant insect cell lines. This is signif- specific genome manipulation in the insect cell lines most commonly icant because these tools will enable new efforts to enhance used as hosts in the BICS. These cell lines include Sf9 (8) and High the capabilities and expand the utility of this important Five (9), which are derived from the lepidopteran insects Spodoptera protein production platform. frugiperda (Sf) and Trichoplusia ni (Tn), respectively. Sf9 and High Five cells clearly have the machinery required for Author contributions: H.M.-A. designed research; H.M.-A. performed research; H.M.-A. protein N-glycosylation, but cannot synthesize the same end and D.L.J. analyzed data; and H.M.-A. and D.L.J. wrote the paper. products as mammalian cells (reviewed in refs. 10–14). More Conflict of interest statement: D.L.J. is the President of and H.M.-A. was a consultant for GlycoBac, LLC. D.L.J. and H.M.-A. are coinventors on a US provisional patent application specifically, both of these lepidopteran insect cell lines can transfer based on the work described herein. N-glycan precursors to nascent polypeptides and trim them to This article is a PNAS Direct Submission. V.M.P. is a guest editor invited by the Editorial produce processing intermediates identical to those produced by Board. mammalian cells. However, neither can process those intermedi- 1Present address: Department of Nutrition and Food Science, Graduate School of Human- ates through the additional steps needed to produce larger, ities and Sciences, Ochanomizu University, Tokyo 112-8610, Japan. mammalian-like structures with terminal sialic acids. Interestingly, 2To whom correspondence should be addressed. Email: [email protected]. we know insect cells encode the machinery needed to produce This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sialylated N-glycans and we also know some cells in whole 1073/pnas.1705836114/-/DCSupplemental. 9068–9073 | PNAS | August 22, 2017 | vol. 114 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1705836114 Downloaded by guest on October 2, 2021 nuclease assays on puromycin-resistant derivatives. The results of this control experiment showed the Dm-fdl gene was efficiently edited in S2R+ cells transfected with the DmU6 vector encoding the Dm-fdl–specific sgRNA and in S2R+ cells transfected with AcCas9DmFDLt3, a previously described CRISPR-Cas9 vector encoding a Dm-fdl–specific sgRNA (28), but not in S2R+ cells transfected with a vector encoding Cas9 alone (Fig. S1B). Sim- ilarly, the Bm-fdl gene was efficiently edited in BmN cells transfected with each of three BmU6-2 vectors encoding differ- ent Bm-fdl–specific sgRNAs, but not in BmN cells trans- fected with a vector encoding Cas9 alone (Fig. S1D). These results indicated our CRISPR-Cas9 vectors produced func- tional Cas9 under ie1 promoter control, functional sgRNAs under DmU6:96Ab and BmU6-2 promoter control, and also showed they Fig. 1. D. melanogaster and B. mori U6 promoters do not support CRISPR- could be used for efficient CRISPR-Cas9 editing of endogenous Cas9 editing in Sf9 cells. (A) Diagram showing generic CRISPR-Cas9 vectors gene targets in cells from the homologous species. encoding, (Left to Right) SpCas9 under the control of a baculovirus ie1 pro- Therefore, we constructed DmU6:96Ab and BmU6-2 moter, an sgRNA expression cassette that includes an insect species-specific CRISPR-Cas9 vectors encoding sgRNAs with three different U6 promoter and a targeting sequence cloning site consisting of two SapI Sf-fdl targeting sequences (Fig. 1B and Table S1) and used them recognition sites, and a puromycin-resistance marker under the control of to transfect Sf9 cells in an effort to edit the Sf-fdl gene. However, baculovirus hr5 enhancer and ie1 promoter elements. (B) Diagram showing Sf- fdl gene structure and highlighting specific Cas9 targeting sequences (Table S1) CEL-I nuclease assays revealed no evidence of Sf-fdl indels in and PCR primer sites. (C) CEL-I nuclease assay results obtained using genomic the resulting puromycin-resistant Sf9 derivatives (Fig. 1C). Be- DNA from Sf9 cells edited with CRISPR-Cas9 vectors encoding various Sf-fdl cause the results obtained with D. melanogaster and B. mori cells targeting sequences (SfFDLt1, SfFDLt2, and SfFDLt3) (Table S1) under the con- indicated these vectors induced adequate Cas9 and pac expres- trol of either the DmU6:96Ab or the BmU6-2 promoter. sion, this result suggested the DmU6 and BmU6 promoters were unable to support adequate sgRNA expression in Sf9 cells, which are derived from a heterologous insect species. Therefore, we biological systems, including insect cell systems. In fact, it has concluded we needed to identify an endogenous SfU6 promoter been shown that endogenous U6 promoters can be used to drive to induce sgRNA expression in Sf9 cells.
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