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      Trimming the genomic fat: minimising and re-functionalising genomes using synthetic biology

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          Abstract

          Naturally evolved organisms typically have large genomes that enable their survival and growth under various conditions. However, the complexity of genomes often precludes our complete understanding of them, and limits the success of biotechnological designs. In contrast, minimal genomes have reduced complexity and therefore improved engineerability, increased biosynthetic capacity through the removal of unnecessary genetic elements, and less recalcitrance to complete characterisation. Here, we review the past and current genome minimisation and re-functionalisation efforts, with an emphasis on the latest advances facilitated by synthetic genomics, and provide a critical appraisal of their potential for industrial applications.

          Abstract

          Naturally evolved genomes tend to be unnecessarily large and redundant, and are not optimised for biotechnological or research applications. In this review, the authors explore genome minimization and re-functionalisation approaches, and discuss their potential for industrial applications.

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          Most cited references65

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          Search-and-replace genome editing without double-strand breaks or donor DNA

          Summary Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2–5 . Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct up to 89% of known genetic variants associated with human diseases.
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            Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.

            We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.
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              Creation of a bacterial cell controlled by a chemically synthesized genome.

              We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.
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                Author and article information

                Contributors
                xin.xu@mq.edu.au
                tom.williams@mq.edu.au
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                8 April 2023
                8 April 2023
                2023
                : 14
                : 1984
                Affiliations
                [1 ]GRID grid.1004.5, ISNI 0000 0001 2158 5405, ARC Centre of Excellence in Synthetic Biology and School of Natural Sciences, , Macquarie University, ; Sydney, NSW 2109 Australia
                [2 ]GRID grid.4563.4, ISNI 0000 0004 1936 8868, School of Life Sciences, , University of Nottingham, ; Nottingham, NG7 2RD UK
                [3 ]GRID grid.7445.2, ISNI 0000 0001 2113 8111, Imperial College Centre for Synthetic Biology, , Imperial College London, ; London, SW7 2AZ UK
                [4 ]GRID grid.7445.2, ISNI 0000 0001 2113 8111, Department of Bioengineering, , Imperial College London, ; London, SW7 2AZ UK
                [5 ]GRID grid.10306.34, ISNI 0000 0004 0606 5382, Wellcome Trust Sanger Institute, ; Cambridgeshire, CB10 1SA UK
                Author information
                http://orcid.org/0000-0002-5644-5397
                http://orcid.org/0000-0003-3910-4782
                http://orcid.org/0000-0001-9127-3175
                http://orcid.org/0000-0001-5392-976X
                http://orcid.org/0000-0001-9015-9418
                http://orcid.org/0000-0002-0594-3441
                Article
                37748
                10.1038/s41467-023-37748-7
                10082837
                37031253
                8883ecdc-4920-477c-9454-a164a7a87261
                © The Author(s) 2023

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 17 November 2022
                : 30 March 2023
                Funding
                Funded by: ARC Centre of Excellence in Synthetic Biology
                Categories
                Review Article
                Custom metadata
                © The Author(s) 2023

                Uncategorized
                synthetic biology,biosynthesis
                Uncategorized
                synthetic biology, biosynthesis

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