Construction of a minimal genome as a chassis for synthetic biology

2016 ◽  
Vol 60 (4) ◽  
pp. 337-346 ◽  
Author(s):  
Bong Hyun Sung ◽  
Donghui Choe ◽  
Sun Chang Kim ◽  
Byung-Kwan Cho
Keyword(s):  
Synthetic Biology ◽  
Genome Stability ◽  
Large Scale ◽  
Genome Engineering ◽  
Essential Gene ◽  
Industrial Applications ◽  
Microbial Genomes ◽  
Gene Set ◽  
Minimal Genome ◽  
A Genome

Microbial diversity and complexity pose challenges in understanding the voluminous genetic information produced from whole-genome sequences, bioinformatics and high-throughput ‘-omics’ research. These challenges can be overcome by a core blueprint of a genome drawn with a minimal gene set, which is essential for life. Systems biology and large-scale gene inactivation studies have estimated the number of essential genes to be ∼300–500 in many microbial genomes. On the basis of the essential gene set information, minimal-genome strains have been generated using sophisticated genome engineering techniques, such as genome reduction and chemical genome synthesis. Current size-reduced genomes are not perfect minimal genomes, but chemically synthesized genomes have just been constructed. Some minimal genomes provide various desirable functions for bioindustry, such as improved genome stability, increased transformation efficacy and improved production of biomaterials. The minimal genome as a chassis genome for synthetic biology can be used to construct custom-designed genomes for various practical and industrial applications.

scholarly journals Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis

2021 ◽  
Vol 12 ◽  
Author(s):  
Aihua Deng ◽  
Zhaopeng Sun ◽  
Tiantian Wang ◽  
Di Cui ◽  
Lai Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Synthetic Biology ◽  
High Throughput Screening ◽  
Large Scale ◽  
Genome Engineering ◽  
Natural Transformation ◽  
Efficient Design ◽  
Accelerated Evolution ◽  
One Step ◽  
Tyrosine Biosynthesis

Multiplex engineering at the scale of whole genomes has become increasingly important for synthetic biology and biotechnology applications. Although several methods have been reported for engineering microbe genomes, their use is limited by their complex procedures using multi-cycle transformations. Natural transformation, involving in species evolution by horizontal gene transfer in many organisms, indicates its potential as a genetic tool. Here, we aimed to develop simultaneous multiplex genome engineering (SMGE) for the simple, rapid, and efficient design of bacterial genomes via one-step of natural transformation in Bacillus subtilis. The transformed DNA, competency factors, and recombinases were adapted to improved co-editing frequencies above 27-fold. Single to octuplet variants with genetic diversity were simultaneously generated using all-in-one vectors harboring multi-gene cassettes. To demonstrate its potential application, the tyrosine biosynthesis pathway was further optimized for producing commercially important resveratrol by high-throughput screening of variant pool in B. subtilis. SMGE represents an accelerated evolution platform that generates diverse multiplex mutations for large-scale genetic engineering and synthetic biology in B. subtilis.

scholarly journals Improved sgRNA design in bacteria via genome-wide activity profiling

2018 ◽  
Author(s):  
Jiahui Guo ◽  
Tianmin Wang ◽  
Changge Guan ◽  
Bing Liu ◽  
Cheng Luo ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Large Scale ◽  
Genome Engineering ◽  
Prediction Models ◽  
Prokaryotic Genome ◽  
Model Organism ◽  
Bacterial Cells ◽  
Highly Active ◽  
Promising Tool ◽  
A Genome

AbstractCRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (~70,000 sgRNAs) with Cas9 or its specificity-improved mutant in E. coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified ‘resistant’ genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate the design of CRISPR/Cas9-based genome engineering or screenings in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

scholarly journals Inducible Plasmid Self-Destruction (IPSD) assisted genome engineering in lactobacilli and bifidobacteria

2019 ◽  
Author(s):  
Fanglei Zuo ◽  
Zhu Zeng ◽  
Lennart Hammarström ◽  
Harold Marcotte
Keyword(s):  
Synthetic Biology ◽  
Homologous Recombination ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Knock Out ◽  
Recombination System ◽  
A Genome ◽  
Selection Of

ABSTRACTGenome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria but still, have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called Inducible Plasmid Self-Destruction (IPSD) was successfully used to perform gene knock-out and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.

scholarly journals A universal, genome-wide guide finder for CRISPR/Cas9 targeting in microbial genomes

2017 ◽  
Author(s):  
Michelle Spoto ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Microbial Genomes ◽  
Genome Wide ◽  
Cas9 Protein ◽  
User Friendly

AbstractBackgroundThe CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression or activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, no pan-bacterial, genome-wide tools exist for guide discovery. We have created Guide Finder: a customizable, user-friendly program that can design guides for any annotated bacterial genome.ResultsGuide Finder designs guides from NGG PAM sites for any number of genes using an annotated genome and fasta file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, a feature unique to Guide Finder. Guide Finder has been tested on a variety of diverse bacterial genomes, on average finding guides for 95% of genes. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species.ConclusionsThrough the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies for a variety of bacterial species.

scholarly journals Large-scale transgenic Drosophila resource collections for loss- and gain-of-function studies

2019 ◽  
Author(s):  
Jonathan Zirin ◽  
Yanhui Hu ◽  
Luping Liu ◽  
Donghui Yang-Zhou ◽  
Ryan Colbeth ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Gene Activation ◽  
Somatic Tissue ◽  
Transcription Start ◽  
Synergistic Activation ◽  
A Genome ◽  
Or Genes ◽  
Genome Scale ◽  
Transgenic Drosophila

ABSTRACTThe Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

scholarly journals Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies

Genetics ◽  
2020 ◽  
Vol 214 (4) ◽  
pp. 755-767 ◽  
Author(s):  
Jonathan Zirin ◽  
Yanhui Hu ◽  
Luping Liu ◽  
Donghui Yang-Zhou ◽  
Ryan Colbeth ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Gene Activation ◽  
Somatic Tissue ◽  
Guide Rnas ◽  
Synergistic Activation ◽  
A Genome ◽  
Or Genes ◽  
Genome Scale ◽  
Transgenic Drosophila

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

scholarly journals CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

2018 ◽  
Author(s):  
Timothy J. de Wet ◽  
Irene Gobe ◽  
Musa M. Mhlanga ◽  
Digby F. Warner
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Essential Gene ◽  
Experimental Models ◽  
Bacterial Gene ◽  
Growth And Survival ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Transcriptional Units

AbstractHigh-throughput essentiality screens have enabled genome-wide assessments of the genetic requirements for growth and survival of a variety of bacteria in different experimental models. The reliance in many of these studies on transposon (Tn)-based gene inactivation has, however, limited the ability to probe essential gene function or design targeted screens. We interrogated the potential of targeted, large-scale, pooled CRISPR interference (CRISPRi)-based screens to extend conventional Tn approaches in mycobacteria through the capacity for positionally regulable gene repression. Here, we report the utility of the “CRISPRi-Seq” method for targeted, pooled essentiality screening, confirming strong overlap with Tn-Seq datasets. In addition, we exploit this high-throughput approach to provide insight into CRISPRi functionality. By interrogating polar effects and combining image-based phenotyping with CRISPRi-mediated depletion of selected essential genes, we demonstrate that CRISPRi-Seq can functionally validate Transcriptional Units within operons. Together, these observations suggest the utility of CRISPRi-Seq to provide insights into (myco)bacterial gene regulation and expression on a genome-wide scale.

scholarly journals Exploiting homologous recombination increases SATAY efficiency for loss- and gain-of-function screening

2019 ◽  
Author(s):  
Agnès H. Michel ◽  
Sabine van Schie ◽  
Andreas Mosbach ◽  
Gabriel Scalliet ◽  
Benoît Kornmann
Keyword(s):  
Homologous Recombination ◽  
Large Scale ◽  
Essential Gene ◽  
Gain Of Function ◽  
Comprehensive Description ◽  
Gene Sets ◽  
Genome Wide ◽  
A Genome ◽  
Bacteria And Fungi ◽  
Anti Fungal

The analysis of large-scale transposon mutant libraries is becoming a method of choice for functional genomics in bacteria and fungi. We previously established SAturated Transposon Analysis in Yeast (SATAY) to uncover genes necessary for growth in any condition in S. cerevisiae (Michel et al., 2017). We present an improved version leveraging homologous recombination to increase transposition efficiency by a factor 10, allowing a single experimenter to rapidly perform several parallel screens. We demonstrate its potential by presenting (1) a comparison of the essential gene sets between two yeast laboratory backgrounds, (2) a comprehensive description of essential genes displaying phenotypic delays – we highlight their common features and propose plausible explanations for this phenomenon –, (3) a genome-wide analysis of loss- and gain-of-function mutations conferring sensitivity or resistance to a compendium of 9 anti-fungal compounds. This study highlights the power of this improved SATAY protocol for yeast functional- and pharmaco-genomics.

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

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