key: cord-0795216-mwiyvhp8 authors: Thao, Tran Thi Nhu; Labroussaa, Fabien; Ebert, Nadine; Jores, Joerg; Thiel, Volker title: In-Yeast Assembly of Coronavirus Infectious cDNA Clones Using a Synthetic Genomics Pipeline date: 2020-05-11 journal: Coronaviruses DOI: 10.1007/978-1-0716-0900-2_13 sha: 2f4bf72479f75c20881402dfeffb9e3d6408fe46 doc_id: 795216 cord_uid: mwiyvhp8 The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV). Reverse genetics platforms for viruses allow the generation of infectious viral cDNA clones, and hence the reconstitution of corresponding viruses and their mutants for characterization studies to gain insights into the basis of viral replication and pathogenesis and to foster vaccine development. However, coronavirus reverse genetics is challenging due to their relatively large genome sizes and the instability of certain viral sequences when propagated in bacterial hosts. Accordingly, coronavirus reverse genetics makes use of unconventional methods such as in vitro ligation of cDNA fragments [1] , vaccinia virus as a vector [2, 3] or cloning cDNA as bacterial artificial chromosome (BAC) in E. coli [4] . Constant epidemic/pandemic threats by (re-)emerging RNA viruses necessitate a fast and easy-to-implement reverse genetics platform that enables the reliable assembly of viral genomes in order to rapidly rescue viruses and their mutants for characterization studies. Here, we describe a synthetic genomics-derived assembly platform as a part of a new reverse genetics system for coronaviruses (Fig. 1) . It uses the transformation-associated recombination (TAR) cloning strategy that exploits the inherently robust homologous recombination system of the yeast Saccharomyces cerevisiae (S. cerevisiae) to generate and maintain full-length viral cDNAs as Fig. 1 A schematic workflow of coronavirus reverse genetics system. Yeastbased TAR cloning, a synthetic genomics-derived assembly platform, is employed to assemble full-length viral cDNAs which are maintained as yeast artificial chromosomes (YACs). Yeast clones carrying correctly assembled viral genomes are identified via PCR-based screening of assembly junctions. Reconstitution of viruses starts with in vitro transcription of sequence-confirmed YACs to generate full-length viral mRNA bearing authentic 5 0 -end cap and 3 0 -end poly (A) tail. The viral mRNA is delivered to appropriate mammalian cells via electroporation to allow the recovery of infectious viruses yeast artificial chromosomes (YACs). This method, originally used to isolate eukaryotic DNA fragments in yeast [5] , was later adapted for the construction of large double-stranded DNA viruses [6, 7] as well as entire bacteria genomes (Mycoplasmas,~1 Mb) [8] [9] [10] . Compared to other stepwise assembly methods, TAR cloning simplifies the accurate assembly of virtually any full-length coronavirus cDNA within a single yeast transformation event. It requires co-transformation of overlapping DNA fragments into yeast, irrespective of their sizes and/or their incompatibility with other intermediate hosts. Applied to coronaviruses, this significantly reduces the timeframe required to build infectious clones and rescue recombinant viruses. In addition, it opens up the possibility to simultaneously modify different viral genomic sequences of interest, thus providing a more versatile and rapid pipeline for genome engineering. An equally noteworthy characteristic is the easy establishment of this method in different lab settings, for it does not require any special equipment or infrastructure. The TAR cloning method makes use of the natural ability of S. cerevisiae to recombine overlapping DNA fragments via homologous recombination. In addition to a centromere sequence and a yeast selectable marker, the TAR vector also contains two targeting sequences called "hooks" at both ends overlapping with the 3 0 and 5 0 ends of the viral DNA sequences. The recombination between the TAR vector and its respective homologous sequences after yeast transformation will result in a circular YAC which is able to freely replicate and segregate in the yeast. Free-end viral DNA fragments and a TAR vector should be properly designed and generated to contain appropriate overlaps for efficient in-yeast homologous recombination (Fig. 2 ). 1. Apply a 32-square sticker to an SD-His agar plate. 2. Use a 20 μl pipette tip to pick a single isolated yeast colony and transfer it onto the plate, filling the area of a sticker-divided square. Repeat until 32 colonies have been picked per construct (see Notes 12-14). 3. Incubate agar plates for 1-2 days at 30 C. The GC prep, or Chelex100 preparation, is a fast and easy method to extract yeast genomic DNA and was adopted from a recent publication [13] . This method ensures that the quality and yield of extracted yeast DNA will suffice for PCR-based screening. 1. From 1 cm 2 patch, use a 20 μl pipette tip to collect yeast cells (see Note 14). 2. Resuspend yeast cells in a 1.5-ml microtube containing 100 μl of 5% Chelex100 solution and glass beads. 3. Vortex at high speed for 4 min at room temperature. 4. Heat the mixture at 100 C for 2 min. 5. Centrifuge at 15,000 Â g for 1 min at room temperature. 6. Carefully transfer 50 μl of the supernatant to a new microtube without disturbing the pellet. Yeast transformants carrying the correctly assembled genome can firstly be identified via PCR-based screening that targets the presence of a specific DNA sequence in the final construct, i.e., a sub-fragment of one of the PCR fragments via simplex PCR. 3. Analyze the PCR products on 1% agarose gel (see Note 17). This section describes the preparation of TAR plasmids containing cloned viral cDNAs in preparative amounts to provide sufficient template for in vitro transcription reactions (see Subheading 3.5). The below protocol is basically a midi plasmid preparation; yet it can easily be scaled up if necessary. Additionally, it is important to note that yeast genomic DNA will be extracted alongside the TAR plasmids. 1. Inoculate 200 ml of SD-His medium with a yeast pre-culture containing the TAR clone of interest (see Note 10) . The yeast doubling time should be estimated for the pre-culture (usually 2-to 3-h doubling time). Based on this, the inoculum can be adjusted to the amount of yeasts that will result in an OD 600nm of~2 within 12-16 h. 11. Apply the supernatant to the filter. Allow the column to empty by gravity. 12. Apply 5 ml of equilibration buffer EQU onto the rim of the filter to wash away any applied supernatant that is remaining in the filter (see Note 21). 13. Remove the filter prior to applying wash buffer WASH to avoid low purity. 14. Add 8 ml of wash buffer WASH. 15. Meanwhile, warm up elution buffer ELU at 50 C. 16. Add 5 ml of pre-warmed buffer ELU (50 C) to elute DNA into a new tube. 17. Add 3.5 ml of room-temperature isopropanol to the eluate to precipitate DNA. 18. Mix thoroughly, but avoid vortexing. 19. Centrifuge at 24,000 Â g for 30 min at 4 C. 20. Carefully discard the supernatant without disturbing DNA pellet. 21. Add 2 ml of room-temperature 70% ethanol to wash the DNA pellet. 22. Centrifuge at 24,000 Â g for 15 min at room temperature. 23. Carefully discard the ethanol completely. Allow the DNA pellet to dry at room temperature (see Note 22). 24. Dissolve DNA pellet in appropriate amount (depending on DNA pellet size) of TE buffer or nuclease-free water (see Notes 23 and 24). DNA concentrations can be expected in the range of 50-100 ng/μl in a 50 μl volume. In this protocol, the reconstitution of infectious viruses starts with generating an mRNA encoding the viral N gene and a full-length viral RNA bearing authentic 5 0 -end cap and 3 0 -end poly(A) tail via in vitro transcription. Subsequently, the RNAs are delivered to mammalian cells via electroporation. Once the transfected fulllength viral RNA is translated to produce coronavirus replicase, the virus replication cycle is initiated. For coronaviruses, it has been shown that co-transfection of full-length RNA and N gene RNA helps to increase the rescue efficiency [1] . The protocol below outlines the rescue procedure for mouse hepatitis virus (MHV) and can be adapted when applied to other coronaviruses, especially in terms of target cell lines. 7. Leave DNA to precipitate at À20 C for 30 min. 8. Centrifuge at 16,000 Â g for 30 min at 4 C. Carefully remove the supernatant to avoid disturbing DNA pellet. 9. Wash DNA pellet with 70% ethanol. 10. Centrifuge at 16,000 Â g for 5 min at room temperature. 11. Completely remove the supernatant. Allow DNA pellet to dry at room temperature. 12. Dissolve DNA in 10-20 μl of nuclease-free water. 13. Set up a 50-μl in vitro transcription reaction using the T7 RiboMax™ Large Scale RNA Production System with m 7 G (5 0 )ppp(5 0 )G RNA Cap Structure Analog (see Table 1 ). 14. Incubate at 30 C for 3 h. 2. On day 2, collect all BHK-21 cells in a 50-ml tube by trypsinizing and centrifuging at 430 Â g for 5 min at 4 C. 3. Resuspend cell pellet in 10 ml of ice-cold phosphate-buffered saline (PBS). Ensure that cells are well separated and determine the cell count. 1. This plasmid derives from the pCC1BAC plasmid (Epicenter) and has been modified for the purpose of DNA isolation in yeast using the TAR cloning method. As it stands, the pCC1BAC-his3 is a yeast/E. coli shuttle vector containing bacterial artificial chromosome (BAC) and yeast centromeric plasmid (YCp) sequences for efficient replication in both organisms. It also contains a histidine selectable marker and a centromere (CEN) to be maintained in yeast as a yeast artificial chromosome (YAC). 2. This yeast strain is highly transformable. S. cerevisiae is grown in rich YPD media supplemented with adenine (YPDA). Yeast transformed with the pCC1BAC-his3 is first plated on minimal synthetic defined (SD) agar media without histidine (SD-His). Yeast colonies are subsequently propagated in SD-His broth at 30 C under agitation at 200 rpm. Yeast culture reaching an optical density at 600 nm (OD 600nm ) of~2 is aliquoted in cryovials containing glycerol (15% final concentration) or, e.g., Roti ® -Store yeast cryovials (Carl Roth), and stored at À80 C. 5. In case of the rescue strategy described here, a T7 promoter sequence and a poly (A) tail followed by a unique restriction site are introduced upstream of the 5 0 -UTR and downstream of the 3 0 -UTR of the viral genome, respectively. In addition, the TAR vector pCC1BAC contains a T7 promoter sequence which is removed after PCR amplification of the vector. 6. To minimize the likelihood of introducing undesired mutations, the amplification of input DNA fragments and TAR vector should be performed using high-fidelity polymerases according to the manufacturer's recommendations. 7. Polyacrylamide gel electrophoresis (PAGE) purification is generally necessary for long oligonucleotides (more than 50 bases) and critical 5 0 sequences. 8. If no unspecific PCR amplifications are observed, a PCR cleanup is sufficient. 9. Concentration of each DNA fragment and TAR vector should not be too low to keep the volume of input DNA (overlapping DNA fragments, TAR vector, and DNA carrier) to around 55 μl during yeast transformation, i.e., less than 10% of the final volume of a transformation reaction (see Subheading 3.2, step 2). 10. When starting from a À80 C glycerol stock, it is recommended to start a pre-culture in 10 ml of SD-His broth. 11. The final volume of yeast culture at this step is calculated based on the number of transformation conditions that one is planning to perform. In general, use 3 ml of yeast culture (OD 600nm~1 ) per condition. 12. Use 2 μl of sterile water or SD-His broth for easy streaking of yeast cells on agar surface. 13. If yeast colonies are not well isolated, restreaking should be performed on 12-sector agar plates to produce isolated colonies. 14. Alternatively, 100 μl of an overnight saturated yeast culture can be used. 15. The 10Â primer mix can contain up to 25 primer pairs. 16. To ensure assay performance, the primer mix should be stored at À20 C in small aliquots to avoid multiple cycles of freezing and thawing. 17. Depending on the size difference of generated PCR products, adjust the agarose gel percentage accordingly. 18. The amount of each ingredient can be adjusted; however, the ratio Buffer RES:Zymolyase:β-Mercaptoethanol ¼ 100:10:1 should be maintained. 19. Depending on specific preferences and settings of laboratories, other plasmid preparation kits can also be used, in which case comparable adjustments should be considered and included to ensure optimal plasmid yields. 20. If the supernatant is not yet clear, transfer it to a new tube and repeat centrifugation, preferably at a higher speed if possible. 21. As mentioned in the kit's user manuals, failing to include this step or direct pouring of buffer EQU inside of the filter may result in lower plasmid yield. 22. DNA pellet should not be overdried as it will be more difficult to be dissolved. 23. Vortexing or pipetting with narrow tips to resuspend DNA pellet is not recommended as it causes DNA shearing. 24. DNA should be left at 4 C for several days to be completely dissolved, and subsequently stored at À20 C for long-term usage. 25. To avoid the possibility of degrading RNA in the following steps, it is strongly recommended that assay performers always wear gloves, and thoroughly spray working areas and pipettes with RNase AWAY solution or similar. 26. BHK-21 cells that stably express the N protein can be used if available, and, in this case, N RNA should be omitted in Subheading 3.5.2. Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus Reverse genetics system for the avian coronavirus infectious bronchitis virus reverse genetics system for the avian coronavirus infectious bronchitis virus Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome Specific cloning of human DNA as yeast artificial chromosomes by transformationassociated recombination Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods Cloning, assembly, and modification of the primary human cytomegalovirus isolate Toledo by yeast-based transformation-associated recombination Complete chemical synthesis, assembly, and cloning of a mycoplasma genitalium genome One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic mycoplasma genitalium genome Creation of a bacterial cell controlled by a chemically synthesized genome creation of a bacterial cell controlled by a chemically synthesized genome A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3 0 end target sequence GC preps: fast and easy extraction of stable yeast genomic