Directed genome engineering for genome optimization

D’Halluin, Kathleen; Ruiter, R.K.

doi:10.1387/ijdb.130217kd

Cited by 18 publications

(8 citation statements)

References 58 publications

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“…In contrast, HR, a more precise mode of DNA repair, is preferred for GT [ 12 , 13 ]. Gene targeting through HR requires simultaneous introduction of the nuclease to create targeted DSB at desired genomic location, and donor DNA containing flanking homologies, acting as a template for repair of the DSB [ 21 ].…”

Section: Gene Targeting: a Byproduct Of Genomic Double-strand Breakmentioning

confidence: 99%

Gene targeting and transgene stacking using intra genomic homologous recombination in plants

Kumar¹,

Barone²,

Smith³

2016

Plant Methods

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Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

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Section: Gene Targeting: a Byproduct Of Genomic Double-strand Breakmentioning

confidence: 99%

Gene targeting and transgene stacking using intra genomic homologous recombination in plants

Kumar¹,

Barone²,

Smith³

2016

Plant Methods

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“…Thus, the development and expansion of a biotechnology toolbox (set of various techniques for inserting, adding, removing or editing genes) for C. reinhardtii represent a powerful and major advance for genome engineering and other biotechnology applications for Chlamydomonas and potentially for other green microphytes [4]. The knock-in involves the one-for-one substitution of DNA sequence information in a specific genetic locus or the insertion of DNA sequence not found previously within the locus.…”

Section: Introductionmentioning

confidence: 99%

Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas

Ghribi

Nouemssi

Meddeb-Mouelhi

et al. 2020

Life

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Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.

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“…One of the core principles behind synthetic biology is the rational engineering of an organism to elicit a modification in phenotype orientated for a specific application (D'Halluin and Ruiter, 2013;Esvelt and Wang, 2013;Mol et al, 2018). The introduction of new genes, allowing for the generation of new proteins within a system, is well documented and has myriad applications.…”

Section: Introductionmentioning

confidence: 99%

Lox’d in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments

Shaw

Serrano

Lluch‐Senar

2020

Preprint

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Rational engineering in synthetic biology requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to ultra-sequencing (TnSeq) which determine single gene essentiality. However, epistatic interactions can rapidly alter these profiles post deletion, leading to the redundancy of these maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of Lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, with ultra-sequencing. When LoxTnSeq was applied to the genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.

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Directed genome engineering for genome optimization

Cited by 18 publications

References 58 publications

Gene targeting and transgene stacking using intra genomic homologous recombination in plants

Gene targeting and transgene stacking using intra genomic homologous recombination in plants

Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas

Lox’d in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments

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