Assembly of ascovirus HvAV-3h long DNA fragment using the Transformation-Associated Recombination (TAR) approach in yeast cells.

Abstract

Background: Synthetic biology is a young but rapidly growing field that allows for assembling long DNA fragments, including complete chromosomes. A key approach for long-DNA assembly is the Transformation Associated Recombination (TAR), which relies on efficient homologous recombination in yeast cells. Recent reports indicate that the TAR method efficiently assembles some human and animal viruses characterized by their large DNA genome size. The application of the TAR method to synthesize long DNA fragments derived from insect viruses is scarce. Therefore, this study aimed to explore the TAR approach for the construction of a long DNA fragment (>44.6 Kb) from the insecticidal Heliothesis virescens ascovirus 3h (HvAV-3h) dsDNA genome to assess the suitability of this approach in genome-wide engineering studies in this family of viruses.

Results: The long DNA fragment assembly process involved three stages: first, we amplified 15 segments of about 2.9-3.2 Kb each via PCR. Next, we recombined these segments through three parallel TAR cycles, producing medium-sized fragments of about 15 Kb. Finally, we assembled these fragments in a single TAR cycle to form a long DNA fragment of about 44.6 kb. We identified some positive clones by colony PCR or restriction digestion pattern. To assess the quality of the assembled DNA fragment, we conducted next-generation sequencing (NGS). A comparative analysis of Sanger sequencing for medium-sized fragments and NGS data from the synthesized long-DNA fragment demonstrated a nearly matched mutation profile, suggesting that the identified mutations and deletions were present at initial synthesis. Both datasets aligned with the reference HvAV-3h strain, revealing three specific nucleotide mutations and three unique mutation regions.

Conclusions: Overall, the in vivo TAR assembly method efficiently assembled a long DNA fragment derived from the ascovirus genome as a template. The process is cost-effective and can be scaled up to synthesize the entire genome for gene functional studies.