scholarly journals CReasPy-cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system

2019 ◽  
Author(s):  
Estelle Ruiz ◽  
Vincent Talenton ◽  
Marie-Pierre Dubrana ◽  
Gabrielle Guesdon ◽  
Maria Lluch-Senar ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Artificial Chromosome ◽  
Bacterial Chromosome ◽  
Cycle Times ◽  
Genetic Elements ◽  
Genetically Engineer ◽  
New Strategy ◽  
Cell Current ◽  
Unique Restriction ◽  
Yeast Genetic

ABSTRACTOver the last decade a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or “cloning”) their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeastSaccharomyces cerevisiaehas been used to clone and engineer genomes from viruses, bacteria and algae. The cloning step requires the insertion of yeast genetic elements within the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-Cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast’s highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome ofMycoplasma pneumoniaeas a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.

scholarly journals Tetracycline Resistome of the Organic Pig Gut

2009 ◽  
Vol 75 (6) ◽  
pp. 1717-1722 ◽  
Author(s):  
Katarzyna A. Kazimierczak ◽  
Karen P. Scott ◽  
Denise Kelly ◽  
Rustam I. Aminov
Keyword(s):  
Resistance Genes ◽  
Metagenomic Library ◽  
Antibiotic Resistance Genes ◽  
Artificial Chromosome ◽  
Mobile Genetic Elements ◽  
Structure Characteristic ◽  
Genetic Elements ◽  
Genes Encoding ◽  
Flanking Regions ◽  
Metagenomic Approach

ABSTRACT The occurrence of genes conferring resistance to tetracyclines in the organic pig gut was assessed through the metagenomic approach. Of 9,000 bacterial artificial chromosome clones analyzed, 10 were identified as carrying the known tet(C), tet(W), and tet(40) genes, as well as novel genes encoding resistance to the tetracyclines minocycline and doxycycline. The latter are different from the known tet genes and are homologous to genes encoding UDP-glucose 4-epimerases, with the domain structure characteristic for these enzymes. The majority of the resistance genes were associated with putative mobile genetic elements. The sequence of a novel 9.7-kb plasmid carrying tet(W) and tet(40) was also identified. Conserved flanking regions identified around the tet(W) and tet(40) genes in our metagenomic library may play a role in genetic exchange of these genes. This is the first report describing the occurrence of tet(40) outside the human intestine. The maintenance of antibiotic resistance genes in apparently antibiotic-free animals is probably due to their presence on mobile genetic elements, the fitness cost of which for the cell is ameliorated during the previous antibiotic selection.

scholarly journals Targeted editing and evolution of engineered ribosomes in vivo by filtered editing

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Felix Radford ◽  
Shane D. Elliott ◽  
Alanna Schepartz ◽  
Farren J. Isaacs
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
23S Rrna ◽  
Translational Efficiency ◽  
Rna Molecules ◽  
Genetic Elements ◽  
A Genome ◽  
Self Splicing

AbstractGenome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

2020 ◽  
Author(s):  
Myeong Uk Kuk ◽  
Sekyung Oh ◽  
Joon Tae Park
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Screening Method ◽  
Artificial Chromosome ◽  
Screening Strategy ◽  
Limited Time ◽  
Screening System ◽  
Time Period ◽  
New Strategy ◽  
Entire Procedure

Abstract Background Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone.Results Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days.Conclusion Overall, our new strategy may provide a new avenue for BAC recombeerining, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.

High-yield and plasmid-free biocatalytic production of 5-methylpyrazine-2-carboxylic acid by combinatorial genetic elements engineering and genome engineering of Escherichia coli

2020 ◽  
Vol 134 ◽  
pp. 109488 ◽  
Author(s):  
Liuyan Gu ◽  
Haibo Yuan ◽  
Xueqin Lv ◽  
Guangsheng Li ◽  
Rigang Cong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carboxylic Acid ◽  
Genome Engineering ◽  
High Yield ◽  
Genetic Elements ◽  
Biocatalytic Production

scholarly journals Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

2020 ◽  
Author(s):  
Myeong Uk Kuk ◽  
Sekyung Oh ◽  
Joon Tae Park
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Screening Method ◽  
Artificial Chromosome ◽  
Screening Strategy ◽  
Limited Time ◽  
Screening System ◽  
Time Period ◽  
New Strategy ◽  
Gain Loss ◽  
Entire Procedure

AbstractRecombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone. Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days. Overall, our new strategy may provide a new avenue for BAC recombeerining, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.

scholarly journals Nonviral genome engineering of natural killer cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabrielle M. Robbins ◽  
Minjing Wang ◽  
Emily J. Pomeroy ◽  
Branden S. Moriarity
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Gene Transfer ◽  
Nk Cells ◽  
Natural Killer ◽  
Genome Engineering ◽  
Cellular Therapy ◽  
Nk Cell ◽  
Cell Based Therapy ◽  
Genetically Engineer

AbstractNatural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.

A new strategy for prenatal diagnosis in a sporadic haemophilia B family

Haemophilia ◽  
2001 ◽  
Vol 7 (4) ◽  
pp. 416-418 ◽  
Author(s):  
M. Acquila ◽  
F. Bottini ◽  
A. Valetto ◽  
D. Caprino ◽  
P. G. Mori ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Haemophilia B ◽  
New Strategy

Guidelines Bring New Strategy in Low-Risk AF

2012 ◽  
Vol 45 (15) ◽  
pp. 12-13
Author(s):  
BRUCE JANCIN
Keyword(s):  
Low Risk ◽  
New Strategy

Closing The Ring: A New Twist to Bacterial Chromosome Condensation

2009 ◽  
Vol 137 (4) ◽  
pp. 598-600
Author(s):  
M THANBICHLER
Keyword(s):  
Chromosome Condensation ◽  
Bacterial Chromosome
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