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Bacterial genome engineering using CRISPR RNA-guided transposases
CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. Th...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10055292/ https://www.ncbi.nlm.nih.gov/pubmed/36993567 http://dx.doi.org/10.1101/2023.03.18.533263 |
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author | Gelsinger, Diego R. Vo, Phuc Leo H. Klompe, Sanne E. Ronda, Carlotta Wang, Harris Sternberg, Samuel H. |
author_facet | Gelsinger, Diego R. Vo, Phuc Leo H. Klompe, Sanne E. Ronda, Carlotta Wang, Harris Sternberg, Samuel H. |
author_sort | Gelsinger, Diego R. |
collection | PubMed |
description | CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ~100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques. |
format | Online Article Text |
id | pubmed-10055292 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
spelling | pubmed-100552922023-03-30 Bacterial genome engineering using CRISPR RNA-guided transposases Gelsinger, Diego R. Vo, Phuc Leo H. Klompe, Sanne E. Ronda, Carlotta Wang, Harris Sternberg, Samuel H. bioRxiv Article CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ~100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques. Cold Spring Harbor Laboratory 2023-03-21 /pmc/articles/PMC10055292/ /pubmed/36993567 http://dx.doi.org/10.1101/2023.03.18.533263 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator. |
spellingShingle | Article Gelsinger, Diego R. Vo, Phuc Leo H. Klompe, Sanne E. Ronda, Carlotta Wang, Harris Sternberg, Samuel H. Bacterial genome engineering using CRISPR RNA-guided transposases |
title | Bacterial genome engineering using CRISPR RNA-guided transposases |
title_full | Bacterial genome engineering using CRISPR RNA-guided transposases |
title_fullStr | Bacterial genome engineering using CRISPR RNA-guided transposases |
title_full_unstemmed | Bacterial genome engineering using CRISPR RNA-guided transposases |
title_short | Bacterial genome engineering using CRISPR RNA-guided transposases |
title_sort | bacterial genome engineering using crispr rna-guided transposases |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10055292/ https://www.ncbi.nlm.nih.gov/pubmed/36993567 http://dx.doi.org/10.1101/2023.03.18.533263 |
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