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WIREs Syst Biol Med
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Accelerated genome engineering through multiplexing

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Throughout the biological sciences, the past 15 years have seen a push toward the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide‐based and nuclease‐based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field. WIREs Syst Biol Med 2016, 8:5–21. doi: 10.1002/wsbm.1319 This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods
Definition of multiplexed genome engineering in this review. (a) In multiplexed genome engineering, multiple mutations (colored ovals) are simultaneously introduced into a single genome (black line). (b) In genome‐scale engineering, each genome receives one specific but different mutation.
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Common vector construction and delivery methods of multiplexed clustered, regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) systems. (a) Multiplexing at vector construction stage. Multiple guide RNA (gRNA) or crRNA sequences can be constructed into one vector by either sequential digestion and ligation or one‐step assembly using customized methods. (b) Multiplexing at gRNA delivery stage. Each gRNA was constructed into one separate vector. Multiple vectors or in vitro transcribed gRNAs can be mixed and simultaneously delivered by microinjection or transfection.
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Different types of nuclease‐mediated multiplexing. (a) Multiallelic modification. One nuclease or nuclease pair cleaves multiple copies of the same site. (b) Multigene disruption. Multiple nucleases/nuclease pairs target different loci across the genome. (c) Chromosomal excision. Two distant cleavages and splicing of the two break points cause the loss of a large chromosome fragment. (d) Chromosomal inversion. Two distant cleavages cause the inversion of a large chromosome fragment. (e) Chromosomal translocation. Two cleavages on two different chromosomes cause the translocation of chromosome arms. (f) Chromosomal duplication (a special case of chromosomal translocation). Two cleavages on two sites of two identical chromosomes result in the duplication of chromosome fragment in between the two cleavage sites. The brown star represents a duplicated gene.
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Nuclease‐mediated multiplexed genome engineering can be achieved using (a) zinc finger nucleases (ZFNs), (b) transcription activator‐like effector nucleases (TALENs), and (c) clustered, regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) (note that elements are not drawn to scale).
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Recombineering‐based multiplexed genome engineering. Mutagenic oligonucleotides recombine with the host genome to cause mutations during DNA replication. Multiple mutagenic oligonucleotides (black and gray lines) can be simultaneously introduced into the genome.
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