Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa

Xu, Zeling; Li, Ming; Li, Yanran; Cao, Huiluo; Lu, Miao; Xu, Zhaochao; Higuchi, Yusuke; Yamasaki, Seiji; Nishino, Kunihiko; Woo, Patrick C. Y.; Xiang, Hua; Yan, Aixin

doi:10.1016/j.celrep.2019.10.006

Cited by 55 publications

(55 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Accroding to recent classification studies, there are seven subtypes (I-A to I-G) in type I CRISPR-Cas system 7,36 . In recent years, the type I-A 37 , I-B 38,39 , I-E 40 , and I-F 41,42 CRISPR-Cas have been used for prokaryotic gene engineering in Sulfolobus islandicus (I-A), Clostridium pasteurianum (I-B), Lactobacillus crispatus (I-E), Zymomonas mobilis (I-F), and Pseudomonas aeruginosa (I-F). Besides, type I-B 43 and type I-E [44][45][46] Cascades can work as transcription repressor in Sulfolobus islandicus (I-B) and Escherichia coli (I-E).…”

mentioning

confidence: 99%

“…Their crRNAs consist of 8-nt 5′ handle for Csy1 and Csy2 binding, 32-nt spacers bound by six copies of Csy3 for target recognition, and 20-nt 3′ hairpin for Csy4 binding and pre-crRNA processing 22 . Recently, type I-F CRISPR-Cas system has been used for genome engineering in Zymomonas mobilis 41 and Pseudomonas aeruginosa 42 . However, there has not been any report on the exploitation of the type I-F or type I-Fv CRISPR-Cas system for genome manipulation application in human cells yet.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

et al. 2020

View full text Add to dashboard Cite

Class 2 CRISPR-Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR-Cas constitutes~60% of all the CRISPR-Cas systems. However, only type I-B and I-E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type IF system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type IF Cas proteins, we activate gene transcription in human cells. In most cases, type IF system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type IF system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type IF CRISPR-Cas in human cells.

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Bacteria that express acrs can tolerate “self-targeting” spacers that, in the absence of CRISPR–Cas inhibition, would otherwise cause lethal genomic cleavage 9 , 24 – 26 . The presence of these self-targeting spacers can therefore be used to identify bacterial genomes that likely encode anti-CRISPR proteins capable of inhibiting their endogenous CRISPR system 12 , 13 , 15 , 27 .…”

Section: Resultsmentioning

confidence: 99%

Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

et al. 2020

View full text Add to dashboard Cite

Many prokaryotes employ CRISPR–Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I–F (Pseudomonas, Pectobacterium, and Serratia), I–E (Pseudomonas and Serratia), and I–C (Pseudomonas). We uncover 11 type I–F and/or I–E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.

show abstract

“…A transferrable and integrative I-F Cascade is developed and is active in the recipient P. aeruginosa host Our previous study has demonstrated that the type I-F CRISPR-Cas system encoded in the P. aeruginosa PA154197 strain is highly active for DNA interference and genome editing (13). Hence, we set out to construct a transferrable I-F Cascade for heterologous applications based on this system.…”

Section: Resultsmentioning

confidence: 99%

A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation

Cao

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered by difficulties of introducing the class-specific, multi-component effectors in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a complete and highly active I-F Cascade in the notoriously recalcitrant and diverse P. aeruginosa genomes by conjugation. The transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than the Cas9 system in diverse genetic backgrounds, including removal of the large (21-kb) integrated cassette with efficiency and simplicity. An advanced λred-I-F system enabled editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an 'all-in-one' I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the mini-CRISPR array expressing desired crRNA in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in non-model isolates of pathogens.

show abstract

Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa

Cited by 55 publications

References 55 publications

Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation

Contact Info

Product

Resources

About