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      Recent advances in genome-scale engineering in Escherichia coli and their applications

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          Abstract

          Owing to the rapid advancement of genome engineering technologies, the scale of genome engineering has expanded dramatically. Genome editing has progressed from one genomic alteration at a time that could only be employed in few species, to the simultaneous generation of multiple modifications across many genomic loci in numerous species. The development and recent advances in multiplex automated genome engineering (MAGE)-associated technologies and clustered regularly interspaced short palindromic repeats and their associated protein (CRISPR-Cas)-based approaches, together with genome-scale synthesis technologies offer unprecedented opportunities for advancing genome-scale engineering in a broader range. These approaches provide new tools to generate strains with desired phenotypes, understand the complexity of biological systems, and directly evolve a genome with novel features. Here, we review the recent major advances in genome-scale engineering tools developed for Escherichia coli, focusing on their applications in identifying essential genes, genome reduction, recoding, and beyond.

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
          Article 0 comments Cited 3414 times – based on 0 reviews     Review now
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            Multiplex genome engineering using CRISPR/Cas systems.

            Le Cong, F. Ran, David T. Cox … (2013)
            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

              Alexis Komor, Yongjoo Kim, Michael Packer … (2016)
              Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. 1,2 Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks. 1,2 Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼15-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
              Article 0 comments Cited 1532 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Junbiao Dai
                Zhuobin Liang
                Journal
                Journal ID (nlm-ta): Eng Microbiol
                Journal ID (iso-abbrev): Eng Microbiol
                Title: Engineering Microbiology
                Publisher: Elsevier
                ISSN (Print): 2097-4280
                ISSN (Electronic): 2667-3703
                Publication date PMC-release: 15 September 2023
                Publication date Collection: March 2024
                Publication date (Electronic): 15 September 2023
                Volume: 4
                Issue: 1
                Electronic Location Identifier: 100115
                Affiliations
                [a ]Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen 518132, China
                [b ]CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics. Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
                [c ]Regenerative Medicine Program, Bellvitge Institute for Biomedical Research (IDIBELL) and Program for Clinical Translation of Regenerative Medicine in Catalonia (P-CMRC), L’ Hospitalet de Llobregat, Barcelona 08908, Spain
                [d ]Faculty of Pharmacy and Food Science, Barcelona University, Barcelona 08028, Spain
                [e ]College of Life Sciences, Northwest A&F University, Shaanxi 712100, China
                [f ]Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China
                Author notes
                Article
                Publisher Item ID: S2667-3703(23)00047-4 Publisher ID: 100115
                DOI: 10.1016/j.engmic.2023.100115
                PMC ID: 11611031
                PubMed ID: 39628784
                SO-VID: 941efe88-fdb0-4c03-bd60-30527adfb83d
                Copyright © © 2023 Published by Elsevier B.V. on behalf of Shandong University.
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 31 May 2023
                Date revision received : 12 September 2023
                Date accepted : 14 September 2023
                Categories
                Subject: Review

                Keywords: genome-scale engineering,e. coli,recombineering,crispr-cas
                Data availability:
                Keywords: genome-scale engineering, e. coli, recombineering, crispr-cas

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