Synthetic Genomes: Design and Construction

Synthetic Genomes

• DNA synthesis technology has progressed to the point that synthesizing entire genomes is now practical.
• Various methods have been developed to synthesize single genes, which have led to the ability to massively edit or write entire genomes from scratch.
• Synthetic genomes can essentially be clones of native sequences.
• Endowing genomes with novel properties has promise for addressing questions about evolution and how genomes are fundamentally wired informationally, metabolically, and genetically.
• Focus is given to techniques and technologies related to how to design, build, and deliver big DNA at the genome scale.
• A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

Introduction

• DNA serves as the carrier of genetic information for all living organisms.
• The study of genetics has greatly accelerated since the discovery of DNA structure in the 1950s.
• DNA provides the template for RNA transcription, coding information for protein translation, and enables transmission of genetic information across generations.
• The ability to read DNA sequence was established in the 1970s, and sequencing throughput scaled up massively with the emergence of next-generation sequencing technologies.
• Approximately 15,000 species in the tree of life were completely or partially sequenced as of April 2018.
• Technology advances in oligonucleotide synthesis, DNA assembly, and genome delivery have revolutionized the field of synthetic genomics.
• Recent synthetic genome projects are reviewed through the lens of the design-build-test-learn paradigm, plus the current status and potential future directions of the field.

Design of Synthetic Genomes

• Designing a synthetic genome involves major consideration.

Distinguishing Synthetic and Native Genomes

• Early genome synthesis studies prioritized designing a sequence nearly identical to the wild type to minimize chances of failure.
• Distinguishing the synthetic product from its natural counterpart is important.
• Cello et al. altered 27 nucleotides in a synthetic poliovirus genome sequence (~7.5 kb) to introduce detectable changes.
• Gibson et al. inserted five watermarks (48–143 bp) to encode tracking information into DNA at locations known to tolerate transposon insertion.
• Smith et al. designed a φX174 bacteriophage genome sequence that matched a published reference sequence for which they did not have physical DNA.

Refactoring Native Genomes

• Refactoring is an aggressive strategy notable in viral and compact genomes selected for small size, a process known as streamlining.
• The T7 bacteriophage genome was partially redesigned to define genetic elements as individual parts that otherwise overlap.
• Disentangling genetic elements facilitates assembly, testing, and dissection of specific functionalities.
• The T7 genome should ideally maintain properties similar to those of the wild type while being easier to manipulate.
• This approach has applications for genetic pathway design to obtain better-controlled systems.

Reducing Redundancy in Native Genomes

• Some DNA elements are redundant or deleterious.
• Cells may not need some genetic features when grown under stress-free or nutrient-rich conditions.
• Researchers reduced the size of the E. coli genome by 7% to 29.7% using different designs.
• Multiple-deletion series strains from Blattner’s group were commercialized by Scarab Genomics leading to improved fitness and performance.
• Smaller synthetic genomes resulted in impaired strain fitness in some cases.
• These E. coli genome reduction studies verified the hypothesis that native genomes could be simplified.

Making Designer Alterations and Adding Elements to Endow New Functions

• Designing eukaryotic synthetic genomes requires consideration of basic biology, industrial potential, and biosafety.
• The Synthetic Yeast Genome Project (Sc2.0) is the first eukaryotic genome synthesis project in the world.
• The ultimate goal of Sc2.0 is to build a synthetic yeast genome that gives wild-type fitness while increasing genome versatility to probe new biological questions regarding gene content, genome structure–function relationships, and evolution.
• Retrotransposons, subtelomeric repeated sequences/genes like COS and PAU, and tRNA genes were removed/relocated to minimize genome instability.
• Pre-mRNA and pre-tRNA introns were removed.
• Stop codon swaps and loxPsym site insertion can endow the synthetic genome with new potential functions.
• Restriction site modifications facilitated synthetic chromosome assembly.
• PCRTags, a watermarking system, introduced synonymous changes into the nucleotide sequence within open reading frames which enables a PCR-based assay for synthetic content.
• An ~8% size reduction and 1.1 Mb of sequence alterations occur due to the synthetic genome.
• Segmental swapping of 30–60 kb allowed for early tests of risky strategic elements of the design.
• All genome synthesis projects to date have started with a known reference sequence and imposed redesigns.
• Additionally, having an accurate and comprehensive genome annotation is equally critical.
• DNA elements present in one subspecies but absent in other subspecies are more likely deletable, at least in stress-free, nutrient-rich conditions.
• Synonymous codon substitution, which affects growth fitness in E. coli, and watermark incorporation, which alters mRNA secondary structure in yeast can lead to design bugs .
• Bugs might reveal aspects of codon usage bias, translation efficiency, ribosome binding, or other biological unknowns.

Building Synthetic Genomes From Scratch

• Synthetic genomes are built from the bottom up using single-stranded oligonucleotides as starting material.
• Automating and miniaturizing oligonucleotide synthesis has enabled significant cost reduction.
• Synthesizing oligos longer than 200 nucleotides with high fidelity is challenging due to the repetitive yield problem.
• Single-stranded oligos are stitched together via various methods into longer dsDNA pieces, then assembled to genome size.
• Synthetic DNA may need building in a different host to achieve higher assembly efficiency.

Oligonucleotide Synthesis

• Researchers started trying to chemically join deoxyribonucleotides in vitro after scientists discovered the structure of DNA in the 1950s.
• Technologies result in higher oligo production per day, mainly when they use microarrays to make thousands of oligos in parallel.
• The chemical synthesis approach is unable to infinitely increase product length with high fidelity and hazardous organic solvents are used during synthesis.
• Terminal deoxynucleotidyl transferase (TdT) is dedicated to DNA synthesis as it uses ssDNA as an initiator to polymerize nucleotides by adding dNTPs stepwise to the 3'-OH group of the initiator.
• It has great potential for chip-based DNA synthesis indicating that TdT can incorporate multiple fluorescent nucleotides and catalyze DNA polymerization up to 8 kb on a surface. mRNA
• TdT can incorporate derivatives to functionalities to DNA, like nuclease resistance.