ArticleNucleic acids research2018
High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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Who cites it
2 citing papers in PubMed.
- Natural and engineered cyclodipeptides: Biosynthesis, chemical diversity, and engineering strategies for diversification and high-yield bioproduction.Engineering microbiology · 2023Review
- Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.Chembiochem : a European journal of chemical biology · 2022Review
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Authors and funding
12 authors.
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Abstract
Selective retrieval of sequence-verified oligonucleotides (oligos) from next-generation sequencing (NGS) flow cells, termed megacloning, promises accurate and reliable gene synthesis. However, gene assembly requires a complete collection of overlapping sense and nonsense oligos, and megacloning does not typically guarantee the complete production of sequence-verified oligos. Therefore, missing oligos must be provided via repetitive rounds of megacloning, which introduces a bottleneck for scaled-up efforts at gene assembly. Here, we introduce the concept of high-depth tiled oligo design to successfully utilize megacloned oligos for gene synthesis. Using acquired oligos from a single round of the megacloning process, we assembled 72 of 81 target Cas9-coding gene variants. We further validated 62 of these cas9 constructs, and deposited the plasmids to Addgene for subsequent functional characterization by the scientific community. This study demonstrates the utility of using sequence-verified oligos for DNA assembly and provides a practical and reliable optimized method for high-throughput gene synthesis.
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