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A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
- M. Jinek, Krzysztof Chylinski, Ines Fonfara, Michael H. Hauer, J. Doudna, E. Charpentier
- 17 August 2012
This 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.
Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
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.
The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA
- Ines Fonfara, Hagen Richter, Majda Bratovič, Anaïs Le Rhun, E. Charpentier
- 28 April 2016
It is shown that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference and constitutes the most minimalistic of the CRISPR–Cas systems so far described.
Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases
- Ines Fonfara, U. Curth, A. Pingoud, W. Wende
- Biology, ChemistryNucleic acids research
- 29 September 2011
The 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.
Engineering of temperature- and light-switchable Cas9 variants
Using tsRC9, the first temperature-sensitive Cas9 variant, it is demonstrated that the light sensitivity of paRC9 is specific to the cellular setting, and temperature-dependent transcriptional control over ectopic and endogenous genetic loci is demonstrated.
Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches
It is demonstrated that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage, and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches.
Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase
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 is reported.
Identification of conserved Features of Laglidadg Homing endonucleases
A study of conserved structural features of LAGLIDADG homing endonucleases that might aid further development of methods of broadening the range of cleaved sequences and identifying residues crucial for DNA binding.
Jinek Bacterial Immunity Guided DNA Endonuclease in Adaptive − A Programmable Dual-RNA
This paper presents a meta-analyses of the immune system’s response to chemotherapy and shows clear patterns of decline in response to treatment-side effects of chemotherapy and also investigates the role of immune checkpoints in this response.