High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides

Cho, Namjin; Seo, Han Na; Ryu, Taehoon; Kwon, Euijin; Huh, Sung-Young; Noh, Jinsung; Yeom, Huiran; Hwang, Byung Kook; Ha, Heejeong; Lee, Ji Hyun; Kwon, Sunghoon; Bang, Duhee

doi:10.1093/nar/gky112

Cited by 4 publications

(4 citation statements)

References 29 publications

(31 reference statements)

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“…DNA can be assembled from overlapping oligonucleotides in vitro, including with small-scale high-throughput methods such as microfluidics (Kong et al, 2007;Plesa et al, 2018) or microchips . Megacloning using error-free, microarray-derived oligonucleotides and a high tiling depth was used to produce 72 cas9 genes from different species (Cho et al, 2018); this technique could be used to efficiently generate a library of gene variants to test in metabolic pathways after in silico genome mining. Generation by overlapping oligonucleotides can be problematic where the sequence contains repeats.…”

Section: In Vitro Assemblymentioning

confidence: 99%

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Young

Haines

Storch

et al. 2021

Metabolic Engineering

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Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.

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Section: In Vitro Assemblymentioning

confidence: 99%

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Young

Haines

Storch

et al. 2021

Metabolic Engineering

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“…Over the last decade, several protocols have been developed that allow the assembly of genes and multi‐gene pathways from error‐prone array‐based oligo pools [18,68–70] . They all involve three core steps: First, amplifying sub‐pools of oligos via pool‐specific primers; this is necessary to get enough oligos for the assembly.…”

Section: Synthesizing Individual Genes and Pathwaysmentioning

confidence: 99%

“…Over the last decade, several protocols have been developed that allow the assembly of genes and multi‐gene pathways from error‐prone array‐based oligo pools. [ 18 , 68 , 69 , 70 ] They all involve three core steps: First, amplifying sub‐pools of oligos via pool‐specific primers; this is necessary to get enough oligos for the assembly. Second, assembly of sub‐pools by overlap PCR or ligase chain reaction, and third, an error removal step, that removes erroneous molecules.…”

Section: Synthesizing Individual Genes and Pathwaysmentioning

confidence: 99%

Oligo Pools as an Affordable Source of Synthetic DNA for Cost‐Effective Library Construction in Protein‐ and Metabolic Pathway Engineering

2021

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The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ~350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost � 10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community.

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“…Here we introduce Sniper assembly, a cell-free, low-cost genome synthesis method that utilizes cell-free cloning to synthesize complete phage genomes de novo using low-cost enrichment of sequence-verified microarray-synthesized oligos (Figure C). Sniper assembly was named after the cell-free cloning method Sniper cloning ,, that the method utilizes. Sniper cloning is a high-throughput cell-free cloning system that can retrieve sequence-verified DNA clusters generated on a next-generation sequencing (NGS) substrate (Figure D).…”

mentioning

confidence: 99%

Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides

Yeom

Ryu²,

Lee

et al. 2020

ACS Synth. Biol.

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Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.

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High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides

Cited by 4 publications

References 29 publications

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Oligo Pools as an Affordable Source of Synthetic DNA for Cost‐Effective Library Construction in Protein‐ and Metabolic Pathway Engineering

Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides

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