| 1. |
- Bosley, Katrine S, et al.
(författare)
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CRISPR germline engineering : the community speaks
- 2015
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Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 33:5, s. 478-486
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Bratovic, Majda, et al.
(författare)
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Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches
- 2020
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Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 16:5, s. 587-595
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Tidskriftsartikel (refereegranskat)abstract
- The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering. Tuning CRISPR-Cas9 nuclease specificity enables precision genome engineering. Identifying arginine residues along the bridge helix of SpCas9 that mediate Cas9 mismatch sensitivity enabled engineering of Cas9 with increased specificity in human cells.
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| 3. |
- Broglia, Laura, et al.
(författare)
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An RNA-seq based comparative approach reveals the transcriptome-wide interplay between 3 '-to-5 ' exoRNases and RNase Y
- 2020
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- RNA degradation is an essential process that allows bacteria to control gene expression and adapt to various environmental conditions. It is usually initiated by endoribonucleases (endoRNases), which produce intermediate fragments that are subsequently degraded by exoribonucleases (exoRNases). However, global studies of the coordinated action of these enzymes are lacking. Here, we compare the targetome of endoRNase Y with the targetomes of 3-to-5 ' exoRNases from Streptococcus pyogenes, namely, PNPase, YhaM, and RNase R. We observe that RNase Y preferentially cleaves after guanosine, generating substrate RNAs for the 3 '-to-5 ' exoRNases. We demonstrate that RNase Y processing is followed by trimming of the newly generated 3 ' ends by PNPase and YhaM. Conversely, the RNA 5 ' ends produced by RNase Y are rarely further trimmed. Our strategy enables the identification of processing events that are otherwise undetectable. Importantly, this approach allows investigation of the intricate interplay between endo- and exoRNases on a genome-wide scale.
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| 4. |
- Broglia, Laura, et al.
(författare)
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RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B
- 2018
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Ingår i: RNA Biology. - : Taylor & Francis. - 1547-6286 .- 1555-8584. ; 15:10, s. 1336-1347
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Tidskriftsartikel (refereegranskat)abstract
- Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.
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| 5. |
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| 6. |
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| 7. |
- Charpentier, Emmanuelle, et al.
(författare)
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Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity
- 2015
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Ingår i: FEMS Microbiology Reviews. - : Oxford University Press (OUP). - 0168-6445 .- 1574-6976. ; 39:3, s. 428-441
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Forskningsöversikt (refereegranskat)abstract
- CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.
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| 8. |
- Charpentier, Emmanuelle, et al.
(författare)
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Biotechnology : rewriting a genome
- 2013
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 495:7439, s. 50-51
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 9. |
- Charpentier, Emmanuelle, et al.
(författare)
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Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.
- 1999
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 181:11, s. 3368-3374
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Tidskriftsartikel (refereegranskat)abstract
- We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.
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| 10. |
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