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Fonfara, Ines
Publications (7 of 7) Show all publications
Richter, F., Fonfara, I., Gelfert, R., Nack, J., Charpentier, E. & Möglich, A. (2017). Switchable Cas9. Current Opinion in Biotechnology, 48, 119-126
Open this publication in new window or tab >>Switchable Cas9
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2017 (English)In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 48, p. 119-126Article, review/survey (Refereed) Published
Abstract [en]

Ever since its discovery, Cas9 from Streptococcus pyogenes has revolutionized biology by enabling analysis and engineering of genomes with unprecedented precision and ease. To fine-tune on-target effects and to mitigate adverse effects caused by untimely and off-target action of Cas9, strategies have been developed to control its activity at the post-translational stage via external trigger signals. Control is either achieved by modifying the Cas9 protein itself or its programmable RNA molecules. To date, switchable Cas9 variants responding to small ligands, light or temperature have been engineered. With these variants in hand, the regulation and modification of genomes can be accomplished in graded and ever more precise manner.

Place, publisher, year, edition, pages
CURRENT BIOLOGY LTD, 2017
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-143648 (URN)10.1016/j.copbio.2017.03.025 (DOI)000418313200017 ()28456061 (PubMedID)
Available from: 2018-01-08 Created: 2018-01-08 Last updated: 2018-06-09Bibliographically approved
Richter, F., Fonfara, I., Bouazza, B., Schumacher, C. H., Bratovic, M., Charpentier, E. & Moeglich, A. (2016). Engineering of temperature- and light-switchable Cas9 variants. Nucleic Acids Research, 44(20), 10003-10014
Open this publication in new window or tab >>Engineering of temperature- and light-switchable Cas9 variants
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2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 20, p. 10003-10014Article in journal (Refereed) Published
Abstract [en]

Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via amechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Es-cherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29 degrees C but negligible activity at 37. C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-132323 (URN)10.1093/nar/gkw930 (DOI)000393817800006 ()27744350 (PubMedID)
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-06-09Bibliographically approved
Eggenschwiler, R., Moslem, M., Fráguas, M. S., Galla, M., Papp, O., Naujock, M., . . . Cantz, T. (2016). Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase. Scientific Reports, 6, Article ID 38198.
Open this publication in new window or tab >>Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38198Article in journal (Refereed) Published
Abstract [en]

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-130086 (URN)10.1038/srep38198 (DOI)000389185500001 ()
Available from: 2017-01-13 Created: 2017-01-11 Last updated: 2018-06-09Bibliographically approved
Fonfara, I., Richter, H., Bratovic, M., Le Rhun, A. & Charpentier, E. (2016). The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature, 532(7600), 517-520
Open this publication in new window or tab >>The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA
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2016 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 532, no 7600, p. 517-520Article in journal (Refereed) Published
Abstract [en]

CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA(1). The well-studied types I, II and III utilize a set of distinct CRISPR-associated ( Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)(2-5) and the mature crRNAs then guide a complex of Cas proteins ( Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA(6-12). In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN- 3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang(16). The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V- A constitutes the most minimalistic of the CRISPR- Cas systems so far described.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-120803 (URN)10.1038/nature17945 (DOI)000374815900051 ()
Available from: 2016-05-25 Created: 2016-05-23 Last updated: 2018-06-07Bibliographically approved
Fonfara, I., Le Rhun, A., Chylinski, K., Makarova, K. S., Lécrivain, A.-L., Bzdrenga, J., . . . Charpentier, E. (2014). Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Research, 42(4), 2577-2590
Open this publication in new window or tab >>Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
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2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2577-2590Article in journal (Refereed) Published
Abstract [en]

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-87653 (URN)10.1093/nar/gkt1074 (DOI)000332381000048 ()
Available from: 2014-04-08 Created: 2014-04-07 Last updated: 2018-06-08Bibliographically approved
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821
Open this publication in new window or tab >>A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity
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2012 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 337, no 6096, p. 816-821Article in journal (Refereed) Published
Abstract [en]

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-59521 (URN)10.1126/science.1225829 (DOI)000307535600036 ()
Available from: 2012-09-17 Created: 2012-09-17 Last updated: 2018-06-08Bibliographically approved
Fonfara, I., Curth, U., Pingoud, A. & Wende, W. (2012). Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases. Nucleic Acids Research, 40(2), 847-860
Open this publication in new window or tab >>Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases
2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 2, p. 847-860Article in journal (Refereed) Published
Abstract [en]

Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.

Place, publisher, year, edition, pages
Oxford University Press, 2012
Keywords
zink-finger nuclease, double-strand breaks, pvuII endonuclease, TAL effectors, targeting DNA, homologous recombination, crystal structure, cleavage domain, gene therapy, in vivo
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-93871 (URN)10.1093/nar/gkr788 (DOI)000299095900039 ()
Available from: 2014-10-01 Created: 2014-10-01 Last updated: 2018-06-07Bibliographically approved
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