A catalogue of biochemically diverse CRISPR-Cas9 orthologs.
Giedrius Gasiunas,Joshua K. Young,Tautvydas Karvelis,Darius Kazlauskas,Tomas Urbaitis,Monika Jasnauskaite,Mantvyda M. Grusyte,Sushmitha Paulraj,Po-Hao Wang,Zhenglin Hou,Shane K. Dooley,Mark Cigan,Clara M. Alarcon,N. Doane Chilcoat,Greta Bigelyte,Jennifer L. Curcuru,Megumu Mabuchi,Zhiyi Sun,Ryan T. Fuchs,Ezra Schildkraut,Peter Weigele,William E. Jack,G. Brett Robb,Česlovas Venclovas,Virginijus Siksnys +24 more
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TLDR
Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target.Abstract:
Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases. A few bacterial Cas9 nucleases have been repurposed as genome editing tools. Here, the authors use bioinformatic and biochemical analyses to characterize 79 Cas9 proteins, revealing substantial functional diversity and thus expanding the available toolbox of RNA-programmable CRISPR-associated nucleases.read more
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CRISPR technologies and the search for the PAM-free nuclease
TL;DR: In this article, the authors review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering and address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.
Journal ArticleDOI
The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases
Han Altae-Tran,Soumya Kannan,F. Esra Demircioglu,Rachel Oshiro,Suchita P. Nety,Luke J. McKay,Mensur Dlakić,William P. Inskeep,Kira S. Makarova,Rhiannon K. Macrae,Eugene V. Koonin,Feng Zhang +11 more
TL;DR: IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interact...
Journal ArticleDOI
The application of DNA polymerases and Cas9 as representative of DNA-modifying enzymes group in DNA sensor design (review).
TL;DR: The main mechanisms of DNA metabolism (replication cycle, amplification) and the genomeediting tool CRISPR-Cas9 are purposefully discussed in order to address strategic possibility to design DNA-sensors based on immobilized DNA-enzymes.
Journal ArticleDOI
Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.
Sarah Balderston,Jeffrey Taulbee,Elizabeth Celaya,Kandace Fung,Amanda Jiao,Kasey Smith,Reza Hajian,Giedrius Gasiunas,Simonas Kutanovas,Daehwan Kim,Jonathan Parkinson,Kenneth Dickerson,Juan José Ripoll,Regis Peytavi,Hsiang-Wei Lu,Francie Barron,Brett R. Goldsmith,Philip G. Collins,Irina M. Conboy,Virginijus Siksnys,Kiana Aran,Kiana Aran +21 more
TL;DR: In this article, a single-nucleotide specificity was achieved by using liquid-gated graphene field effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer.
Journal ArticleDOI
Advances and insights in the diagnosis of viral infections.
TL;DR: A comprehensive overview of molecular analytical methods has shown that the assay's sensitivity, accuracy, and suitability for virus detection depends on the choice of the number of regions in the viral open reading frame (ORF) genome sequence and the validity of the selected analytical method as mentioned in this paper.
References
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