Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.

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CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses

Molecular Biology of the Cell ◽

10.1091/mbc.e18-04-0214 ◽

2018 ◽

Vol 29 (20) ◽

pp. 2370-2377 ◽

Cited By ~ 5

Author(s):

Kuan-Chung Su ◽

Mary-Jane Tsang ◽

Neil Emans ◽

Iain M. Cheeseman

Keyword(s):

Dna Cleavage ◽

Human Cells ◽

Target Cells ◽

Gene Products ◽

Guide Rna ◽

Protein Depletion ◽

Functional Studies ◽

Culture Cells ◽

Rna Transfection ◽

Biological Studies

A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.

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A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

Nucleic Acids Research ◽

10.1093/nar/gkx783 ◽

2017 ◽

Vol 45 (19) ◽

pp. 11295-11304 ◽

Cited By ~ 60

Author(s):

Mengjun Tu ◽

Li Lin ◽

Yilu Cheng ◽

Xiubin He ◽

Huihui Sun ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Human Cells ◽

Cleavage Activity ◽

Dna Cleavage Activity

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Engineered Campylobacter jejuni Cas9 variant with enhanced activity

10.21203/rs.3.rs-512474/v1 ◽

2021 ◽

Author(s):

Ryoya Nakagawa ◽

Soh Ishiguro ◽

Sae Okazaki ◽

Hideto Mori ◽

Mamoru Tanaka ◽

...

Keyword(s):

Genome Editing ◽

Campylobacter Jejuni ◽

Genome Engineering ◽

Cytosine Deaminase ◽

Human Cells ◽

Adeno Associated Virus ◽

Cleavage Activity ◽

Protospacer Adjacent Motif ◽

Its Gene

Abstract The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

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RNA-programmed genome editing in human cells

eLife ◽

10.7554/elife.00471 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 1223

Author(s):

Martin Jinek ◽

Alexandra East ◽

Aaron Cheng ◽

Steven Lin ◽

Enbo Ma ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Human Cells ◽

Limiting Factor ◽

Dna Breaks ◽

Genetic Changes ◽

Rna Sequence ◽

Guide Rna ◽

Double Strand Dna Breaks

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.

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DNA-cleavage activity of the iron(II) complex with optically active ligands, meta- and para-xylyl-linked N’,N’-dipyridylmethyl-cyclohexane-1,2-diamine

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2021.127834 ◽

2021 ◽

Vol 36 ◽

pp. 127834

Author(s):

Koichi Kato ◽

Yoshimi Ichimaru ◽

Yoshinori Okuno ◽

Yoshihiro Yamaguchi ◽

Wanchun Jin ◽

...

Keyword(s):

Dna Cleavage ◽

Optically Active ◽

Cleavage Activity ◽

Dna Cleavage Activity

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Antimicrobial Efficacy and Spectrum of Phosphorous-Fluorine Co-Doped TiO2 Nanoparticles on the Foodborne Pathogenic Bacteria Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus

Foods ◽

10.3390/foods10081786 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1786

Author(s):

György Schneider ◽

Bettina Schweitzer ◽

Anita Steinbach ◽

Botond Zsombor Pertics ◽

Alysia Cox ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Campylobacter Jejuni ◽

Food Industry ◽

Pathogenic Bacteria ◽

Antibacterial Activities ◽

Tio2 Nps ◽

E Coli ◽

Foodborne Pathogenic Bacteria ◽

And Staphylococcus Aureus ◽

Co Doped

Contamination of meats and meat products with foodborne pathogenic bacteria raises serious safety issues in the food industry. The antibacterial activities of phosphorous-fluorine co-doped TiO2 nanoparticles (PF-TiO2) were investigated against seven foodborne pathogenic bacteria: Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus. PF-TiO2 NPs were synthesized hydrothermally at 250 °C for 1, 3, 6 or 12 h, and then tested at three different concentrations (500 μg/mL, 100 μg/mL, 20 μg/mL) for the inactivation of foodborne bacteria under UVA irradiation, daylight exposure or dark conditions. The antibacterial efficacies were compared after 30 min of exposure to light. Distinct differences in the antibacterial activities of the PF-TiO2 NPs, and the susceptibilities of tested foodborne pathogenic bacterium species were found. PF-TiO2/3 h and PF-TiO2/6 h showed the highest antibacterial activity by decreasing the living bacterial cell number from ~106 by ~5 log (L. monocytogenes), ~4 log (EHEC), ~3 log (Y. enterolcolitca, S. putrefaciens) and ~2.5 log (S. aureus), along with complete eradication of C. jejuni and S. Typhimurium. Efficacy of PF-TiO2/1 h and PF-TiO2/12 h NPs was lower, typically causing a ~2–4 log decrease in colony forming units depending on the tested bacterium while the effect of PF-TiO2/0 h was comparable to P25 TiO2, a commercial TiO2 with high photocatalytic activity. Our results show that PF-co-doping of TiO2 NPs enhanced the antibacterial action against foodborne pathogenic bacteria and are potential candidates for use in the food industry as active surface components, potentially contributing to the production of meats that are safe for consumption.

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A YoeB toxin cleaves both RNA and DNA

Scientific Reports ◽

10.1038/s41598-021-82950-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Julia McGillick ◽

Jessica R. Ames ◽

Tamiko Murphy ◽

Christina R. Bourne

Keyword(s):

Dna Cleavage ◽

Divalent Cations ◽

Nuclease Activity ◽

Dose Dependence ◽

Cleavage Activity ◽

Double Stranded Dna ◽

Dna Cleavage Activity ◽

Evolutionarily Conserved ◽

Toxin Protein ◽

Rna And Dna

AbstractType II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

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