Synthetic chromosomes, genomes, viruses, and cells

Abstract

Synthetic genomics is the construction of viruses, bacteria, and eukaryotic cells with synthetic genomes. It involves two basic processes: synthesis of complete genomes or chromosomes and booting up of those synthetic nucleic acids to make viruses or living cells. The first synthetic genomics efforts resulted in the construction of viruses. This led to a revolution in viral reverse genetics and improvements in vaccine design and manufacture. The first bacterium with a synthetic genome led to construction of a minimal bacterial cell and recoded Escherichia coli strains able to incorporate multiple non-standard amino acids in proteins and resistant to phage infection. Further advances led to a yeast strain with a synthetic genome and new approaches for animal and plant artificial chromosomes. On the horizon there are dramatic advances in DNA synthesis that will enable extraordinary new opportunities in medicine, industry, agriculture, and research.

Figure 1.. Synthetic Gene Segment Assembly with Error Correction and Rescue of Synthetic Influenza Viruses from a Panel of Backbones.

A. Schematic diagram of the assembly procedure. Error correction reduced the rate from 1 error per 1328 bp to 1 error per 9589. X, sites of oligonucleotide errors. Blue arrow, HA or NA coding sequence; gray, plasmid backbone sequence; green arrow, CMV promoter; purple arrow, human pol I promoter; red arrow, murine pol I terminator; brown arrow, pol II terminator; black rectangle, UTR. B. Schematic diagram of the rescue of synthetic influenza viruses from multiple backbones for types A and B influenza strains. PR8x, derivative of a PR8 strain adapted over 5 passages for growth in MDCK cells; Hes, A/Hessen/105/2007 (H1N1); A/CA, A/California/7/2009 (H1N1); Brisbane, B/Brisbane/60/2008 (Victoria lineage); Panama, B/Panama/45/1990 (Yamagata lineage). The photograph of MDCK cells was made by and used with permission from Benjamin Sievers, JCVI.

Figure 2.. Genome Transplantation.

A. Yeast cells or bacterial cells containing the donor genome to be transplanted are encased in low melt agarose blocks. Yeast cells are spheroplasted using zymolase and digested with proteinase K. B. This leaves the donor genome inside caverns in the agarose, and not sheared during the purification The agarose is melted to gently retrieve the DNA. C. The donor DNA (red) and M. capricolum cells are mixed with polyethylene glycol (PEG) to increase recipient cell membrane fluidity and CaCl₂, to mask the DNA charge, resulting in the donor genome entering the recipient cell (at very low frequency). D. The transiently diploid cells are transferred to growth media and begin to grow and divide. E. After several hours, the cells are treated with tetracycline. Only the cells with the synthetic donor genome containing a tetracycline resistance marker survive.

Figure 3.. Three technologies critical to the construction of the first bacterium with a synthetic genome.

These Synthetic Genomics technologies were developed by the JCVI to enable construction of bacteria with chemically synthesized genomes. Prior to the synthesis of the M. genitalium genome in 2008 (Gibson et al., 2008a), DNA synthesis was used to produce molecules only as large as 32 kb; however the process was slow and inefficient. The genome synthesis technology developed by the JCVI greatly accelerated the process as well as enabling the in vitro synthesis of much larger DNA molecules. Yeast cloning of bacterial genomes was developed both for the final assembly of large overlapping sub-genomic DNA molecules that were transformed into yeast along with a 3–5 kb yeast vector sequence as yeast centromeric plasmids. This enabled parking the synthetic genome in yeast cells so that amount of bacterial genomic DNA needed for genome transplantation could be produced from large amounts of those yeast. Genome transplantation as depicted in Figure 2, boots up the synthetic genome isolated from yeast by installing it in a suitable bacterial recipient cell so that the new genome commandeers the recipient cell to produce a new cell with the genotype and phenotype of the synthetic genome.

Figure 4.. DNA synthesis by electrochemistry.

(A) (Left) Cycle of synthesis, illustrated adding a phosphoramidite to a site: deprotection is driven by localized acid generation at the site. (Right) Localized Acid for deprotection, achieved by decomposition of Hydroquinone (HQ) to release H⁺ acid at the local Working Electrode (WE), and active removal of acid by recombination with a cognate base, oxidized tetrachloro-1,4-benzoquinone (TQ) generated at the local Counter Electrode (CE). (B) DNA synthesis chip. (Left) A CMOS chip device to drive on-chip DNA synthesis. The chip has three sub-arrays of synthesis pixels (SynPixels) of different sizes to illustrate scalability: Banks 1—3 have pixels with footprint (in microns) 2 × 2, 2 × 3 and 30 × 30 respectively. Each array is controlled by row and column driver circuits, which program the pixels for activation, and provide connection to peripheral current monitoring circuits to monitor the electrochemical processes. (Middle) Voltage control of the central Working Electrode (WE) for acid generation and surrounding Counter Electrode (CE) for base generation, is controlled by a transistor switch circuit. (Right) Annotated microscopic image of the CMOS chip die, showing chip size and subarray dimensions. Insets show electron microscope images of the central Platinum WE and common peripheral CE. (C) On-chip scalable 100-mer synthesis. (Upper) Example of localized, controlled synthesis of oligos on the pixel array, spelling out “HELIX”, with synthesis visualized via a fluorescent microscope image of the synthesized oligos labeled by hybridizing to a fluorescently labeled complementary oligo. (Lower Left) Structure of the 100-mer oligo: 86 nucleotide (nt) poly-T and 15 nt complex sequence. Signals from red (Cy5) and green (FAM) labelling oligos hybridized to these segments are shown. Oligo synthesis is seen to be primarily in the annular silicon surface area between the central platinum electrode surrounding platinum counter electrode. (Lower Right) Example of checkerboard pattern synthesis of two different 15 nt sequences, illustrating the ability to sequence independent sequences at each site, along with the current versus time observed during the 30 cycles of synthesis, showing the net electrochemical currents drawn on the array by the Working Electrodes (WE) and Counter Electrodes (CE) (the information and images are through personal communication with Barry Merriman of Avery Digital Data).

Figure 5.. World’s first pig to human heart transplant performed on January 7, 2022.

The patient, Robert Bennett was not a candidate for a human heart transplant or an artificial heart. The xenotransplantation was a compassionate-use case. Bennett lived for two months.