Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity

Abstract

Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (Syn-ViP). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See'm principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering. K E Y W O R D SDNA chemical synthesis, Golden Gate cloning, plant virome, type IIS restriction enzyme, viral infectious clone assembly INTRODUCTIONViruses are relatively simple biological entities, with genomic components whose size is compatible both in terms of technical feasibility and costs with current advances in de novo DNA synthesis. [1,2] Synthetic genomics is becoming commonplace for the study and engineering of animal viruses, especially those with medical interest and human pandemic potential. [3][4][5][6][7] In plant virology, synthetic genomics can help to demonstrate correctness of genomic sequences, rescue of viruses that might not be physically available (e.g., ancient or environmental samples), as well as to accelerate virus reverse genetics, host-virus interaction studies, biological characterization of emerging viruses, or engineering of biotechnological devices. [8][9][10][11] Adoption of this powerful approach for plant virology has nonetheless lagged and is reported only in a handful of studies. [9] The first reports of synthetic genomic approaches in plant virology included the use of oligonucleotides to

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