دورية أكاديمية
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Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology.

التفاصيل البيبلوغرافية
العنوان: Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology.
المؤلفون: Gussak A; Host-Microbe Interactomics, Animal Sciences, Wageningen University, 6708 WD Wageningen, The Netherlands., Ferrando ML; Host-Microbe Interactomics, Animal Sciences, Wageningen University, 6708 WD Wageningen, The Netherlands., Schrama M; Host-Microbe Interactomics, Animal Sciences, Wageningen University, 6708 WD Wageningen, The Netherlands., van Baarlen P; Host-Microbe Interactomics, Animal Sciences, Wageningen University, 6708 WD Wageningen, The Netherlands., Wells JM; Host-Microbe Interactomics, Animal Sciences, Wageningen University, 6708 WD Wageningen, The Netherlands.
المصدر: ACS synthetic biology [ACS Synth Biol] 2023 Sep 15; Vol. 12 (9), pp. 2546-2560. Date of Electronic Publication: 2023 Aug 21.
نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't
اللغة: English
بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 101575075 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2161-5063 (Electronic) Linking ISSN: 21615063 NLM ISO Abbreviation: ACS Synth Biol Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Washington, D.C. : American Chemical Society, c2012-
مواضيع طبية MeSH: Streptococcus suis*/genetics, Humans ; Animals ; Swine ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Technology ; Anti-Bacterial Agents
مستخلص: Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis , utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis , as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.
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فهرسة مساهمة: Keywords: CRISPR-Cas9; Streptococcus suis; genetic engineering; pathogen; persister cells; zoonotic
المشرفين على المادة: 0 (RNA, Guide, CRISPR-Cas Systems)
0 (Anti-Bacterial Agents)
تواريخ الأحداث: Date Created: 20230821 Date Completed: 20230918 Latest Revision: 20230922
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC10510748
DOI: 10.1021/acssynbio.3c00110
PMID: 37602730
قاعدة البيانات: MEDLINE
الوصف
تدمد:2161-5063
DOI:10.1021/acssynbio.3c00110