Synthetic Genomes: Design and Construction

Synthetic Genomes

• DNA synthesis technology has advanced to the point where synthesizing entire genomes is practical.
• Synthetic genomes can be clones of native sequences, but this approach doesn't expand biological knowledge.
• Endowing genomes with novel properties is promising for addressing questions about evolution and genome wiring.
• Understanding the design, building, and delivery of large DNA at the genome scale could lead to designing genomes from scratch.
• The design-build-test-learn paradigm highlights progress in synthetic genome projects and discusses the field's current status and future directions.

Design of Synthetic Genomes

• Designing a synthetic genome is like creating a blueprint for a building and requires consideration.
• Genome designs must align with the capabilities of genome synthesis or engineering for successful completion.
• Historically, researchers designed genomes from simple to complex, categorized into four types.

Distinguishing Synthetic and Native Genomes

• Early genome synthesis studies prioritized sequences nearly identical to wild type to minimize failure risks.
• Distinguishing the synthetic product from its natural counterpart is important, leading to documented strategies.
• Cello et al. altered 27 nucleotides in a synthetic poliovirus genome to introduce detectable changes.
• Gibson et al. inserted five watermarks (48–143 bp sequences) at intergenic positions to encode tracking information.
• Smith et al. designed a qX174 bacteriophage genome sequence matching a published reference sequence without physical DNA.
• In all cases, designs aimed for minimal changes to functional genome sequences, hoping the synthetic version would function in cells.
• These projects demonstrated that nucleotide sequence alone is sufficient for function, challenging the need for unobserved modifications or components.

Refactoring Native Genomes

• Refactoring is an aggressive strategy for synthetic genome design, streamlining viral and compact genomes.
• The T7 bacteriophage genome was partially redesigned to define genetic elements as individual parts.
• The advantage is that different modules can be assembled and tested individually to dissect functionalities.
• The refactored T7 genome should maintain properties similar to the wild type while being easier to manipulate.
• The refactoring approach has applications for genetic pathway design to obtain better-controlled systems.

Reducing Redundancy in Native Genomes

• It can be hypothesized that not all DNA elements in a genome are necessary.
• Some elements are redundant, and others, such as transposable elements, can have short-term deleterious effects.
• Some genetic features may not be needed under stress-free conditions.
• Escherichia coli has been used to test these hypotheses.
• Researchers reduced the E. coli genome size (7% to 29.7%) using different designs.
• Strains with multiple deletions generated were commercialized by Scarab Genomics.
• Claims made regarding fitness and performance improvement, but smaller genomes sometimes resulted in impaired fitness.
• Experiments tested gene essentiality in the E. coli genome. These studies verified that native genomes could be simplified.

Making Designer Alterations and Adding Elements to Endow new Functions

• Eukaryotic genomes are more complex than prokaryotic genomes due to size, regulation and architecture.
• Designing such synthetic genomes requires consideration of many aspects
• The Synthetic Yeast Genome Project (Sc2.0) builds a synthetic yeast genome in the world that powers wild-type fitness while increasing genome versatility to probe new biological questions regarding gene content, genome structure-function relationships, and evolution.
• Notable departures from previous genome synthesis projects during Sc2.0 design
  • Dramatic changes were made to the Saccharomyces cerevisiae genome sequence to teach us new biology
  • Retrotransposons, tRNA genes, and subtelomeric repeated sequences were repositioned or removed to minimize genome instability
  • pre-mRNA and pre-tRNA introns were removed to question their necessity
  • Stop codon swaps and loxPsym site insertion to endow the synthetic genome with functions, enabling modifications
• Restriction site modifications facilitate the assembly of synthetic chromosomes
• A watermarking system called introduced synonymous changes into the nucleotide sequence within open reading frames enable a polymerase chain reaction (PCR)-based assay for synthetic content
• PCRTags facilitate analysis of intermediates in the assembly of synthetic chromosomes, ensuring synthetic sequences are incorporated but native sequences are eliminated
• Synthetic genome has an ~8% size reduction and 1.1 megabases (Mb) of sequence alterations relative - to the native genome (16)
• Used process to build the synthetic genome was segmental swapping of 30-60 kb elements and allows for early tests of risky strategic elements of the design.
• All genome synthesis were started with a known reference and impose some redesign
• Having accurate genome and comprehensive annotation are critical
• Functional elements have been found in genomes and though computational and experimental approach
• Used Experimental transposon-based mutagenesis and gene knockouts to identify gene essentiality in bacteria and yeast
• Genome comparison across subspecies identifies gene essentiality and are more deletable in stress-free, nutrient-rich conditions
• Genome characterization leads to designing a functional genome
• The design bugs are synonymous codon substitution, watermark incorporation, and ectopic transcription binding sites
• mechanisms reveals codon usage bias, translation efficiency, ribosome binding

Building Synthetic Genomes from Scratch

• Synthetic genomes are constructed from the bottom up using single-stranded oligonucleotides.
• Automating and miniaturizing oligonucleotide synthesis has enabled significant cost reduction.
• Synthesizing oligonucleotides longer than 200 nucleotides with high fidelity is challenging
• Longer DNA sequences are produced by stitching together single-stranded oligos into double-stranded DNA pieces
• Built synthetic DNA sometimes requires a different host to achieve higher assembly efficiency

Oligonucleotide Synthesis

• Researchers started chemically joining deoxyribonucleotides in vitro
• Synthetic phosphoramidite chemistry developed in the 1980s
• High-throughput/automated oligo synthesis technologies result in higher oligonucleotide production per day,
• Low yield and high error caused obstacles
• Chemical synthesis unable to increase product length with high fidelity
• Hazardous organic solvents used during synthesis.
• Enzyme-based single stranded DNA syntheses have potential to polymerize oligonucleotides faster and more accurately Among known DNA and RNA synthesis enzymes, terminal deoxynucleotidyl transferase (TdT) is dedicated to DNA synthesis in a template-independent manner
• Bollum first isolated and characterized TdT in the 1960s
• TdT can incorporate multiple fluorescent nucleotides and catalyze DNA polymerization up to 8 kb on a surface
• it is is also capable of incorporating various nucleotide derivatives endow more more functionalities to to DNA

Synthetic Genome Assembly

• A gene or functional unit has hundreds/thousands of base pairs
• Researchers developed assemble multiple oligos into sequences
• Early gene syntheses involved short oligonucleotides and T4 polynucleotide ligase
• Applied PCA and cloning to hierarchically built the genome (cell at al.) Optimization such as gel purification and 55°C ligation before PCA can dramatically increase assemble length

Sequence Feature Influences on The Cost of Gene Synthesis

• Price increases along with length because errors accumulate more
• Extremes effect the price because their base concentration can reduce information
• Homopolymers are more expensive because lead to expansion or contraction of runs
• Repeats in sequence are more expensive due to misassembly
• PCA-based methods include an intrinsic error-correction step and mismatching bases are removed
• Sequences is usually validated by sequencing for verification
• Commercial prices are subject to filtering rules
• Oligonucleotide synthesis reduces synthesis costs

Synthetic Genome Delivery

• Synthetic genomes must be delivered and tested in living systems
• Bacteriophage genomes can be directly introduced into a host
• Two strategies are one-step delivery and stepwise substitution
• One-step delivery- transplanting DNA from bacterial sequences
• Labrousssa uses several bacterial species as genome donors (increasing Distance phylogentically from
• transfer has successfully been demonstrated only by Mollicules
• Genomes were engineered to do what can be done in yeast
• genome isolation step can be removed by cell fusion
• delivering DNA is applicable to yeast

Top-Down Genome Editing

• DNA double-strand breaks boost the efficiency of DNA recombination; this led to discovering DNA-targeting; reagents stimulating DNA
• CRISPR-Cas9 has been developed for genome modification in bacteria, fungi, invertebrates, vertebrates, and plants
• Multiplexing reduces high-throuput, Cas9 to achieve genome editing
• high-throughput CRISPR-Cas9
• high-throughput is limited to multiple identical sequences
• Red system's process increase HR and DNA recombination
• oligo-mediated allelic replacement with MAGE( multiplex automated genome)

Insides from Synthetic Genomes

• Requires designs, new techno developments
• Section focusing can be focused down more than the rest, so the data provided can be beneficial.
• identified series of bugs that affect the cell
• elimination of codons
• yceQ yccY genes are not transcribed

Ethical Considerations

• Provokes ethical conditions due to outside the laboratory
• gene drives- which combine crispr9 to spread changes through wild life
• editing is split into non heritable somatic
• somatic is more disease correctable Germ line is limited to being natural alleles
• discuss governance of somatic

Future Directions, What Is Next?

• Understand biological aspects and benefit from them
• Genome project includes all organisms , plants , humans
• Stimulates progress in tech
• synthesis will be new as well