A
            <i>pyrF</i>
            -Based Efficient Genetic Manipulation Platform in Acinetobacter baumannii To Explore the Vital DNA Components of Adaptive Immunity for I-F CRISPR-Cas

Wu, Shaofang; Xu, Run; Su, Mengjiao; Gao, Can; Liu, Yang; Chen, Yujia; Luan, Guangxin; Xu, Jin; Wang, Rui

doi:10.1128/spectrum.01957-22

Cited by 6 publications

(8 citation statements)

References 54 publications

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“…6 ). A. baumannii ATCC19606, A. baumannii ATCC17978, WT A. baumannii AYE (Δ pyrF ) and its cas mutants (Δ cas1 , Δ cas3 , and Δ csy ) were from a previous study 35 . The AYE mutants TA-in, TA-out, Δ csy4 , and Δ aac3 were constructed in this study, as previously described 35 (also see below).…”

Section: Methodsmentioning

confidence: 99%

“…3 , all oligonucleotides were produced from Tsingke Biological Technology, Beijing, China. The conjugative plasmid pMo130TFR and its derivate pMo130TFRI (has a lacI gene) were from a previous study 35 , and used for gene expression in this study. The creTA genes of A. baumannii WCHA45, LoGeW2-3, and ANC3789 were amplified using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co.,Ltd).…”

Section: Methodsmentioning

confidence: 99%

“…To construct the AYE mutants TA-in and TA-out (Fig. 5b ), wherein the WCHA45 creTA was inserted between the cas3 and csy1 genes or at the original genomic site of the deleted pyrF gene, the creTA genes and the 5′- and 3′-regions (each 1 kb) flanking the target site were separately amplified, and then inserted into predigested (by BamHI and PstI) pMo130TF (a suicidal vector 35 ) using a four-piece Gibson assembly strategy.…”

Section: Methodsmentioning

confidence: 99%

“…2 ) or aac3 (Supplementary Fig. 7 ) from AYE, the 5′- and 3′-flanking sequences (each 1 kb) were amplified and assembled into pMo130TF 35 using the three-piece Gibson assembly strategy. To complement a plasmid copy of aac3 , its native promoter and coding sequence were amplified and inserted into predigested (by BamHI and PstI) pMo130TFR or pCasAb-Apr 36 .…”

Section: Methodsmentioning

confidence: 99%

“…A pyrF -based Efficient Genetic Manipulation Platform 35 was used for gene knock-in or knock-out in A. baumannii AYE. Plasmids used for gene knock-in or knock-out were constructed as described above.…”

Section: Methodsmentioning

confidence: 99%

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Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens

et al. 2023

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CreTA, CRISPR-regulated toxin-antitoxin (TA), safeguards CRISPR-Cas immune systems by inducing cell dormancy/death upon their inactivation. Here, we characterize a bacterial CreTA associating with the I-F CRISPR-Cas in Acinetobacter. CreT is a distinct bactericidal small RNA likely targeting several essential RNA molecules that are required to initiate protein synthesis. CreA guides the CRISPR effector to transcriptionally repress CreT. We further demonstrate a proof-of-concept antimicrobial strategy named ATTACK, which AssociaTes TA and CRISPR-Cas to Kill multidrug resistant (MDR) pathogens. In this design, CRISPR-Cas is programed to target antibiotic resistance gene(s) to selectively kill MDR pathogens or cure their resistance, and when CRISPR-Cas is inactivated or suppressed by unwanted genetic or non-genetic events/factors, CreTA triggers cell death as the last resort. Our data highlight the diversity of RNA toxins coevolving with CRISPR-Cas, and illuminate a combined strategy of CRISPR and TA antimicrobials to ‘ATTACK’ MDR pathogens.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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