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Characterization of the type IV CRISPR-Cas system of aromatoleum aromaticum EbN1
Characterization of the type IV CRISPR-Cas system of aromatoleum aromaticum EbN1
CRISPR-Cas systems provide prokaryotic adaptive immunity against invading agents, which also stimulated the development of indispensable tools in biological research. CRISPR-Cas systems show remarkable diversity and encompass two classes, six types and 16 subtypes. Among these six computationally classified CRISPR-Cas types, type IV CRISPR-Cas systems remained the only one without experimental data. In this study, we provide the first experimental characterization of a type IV CRISPR-Cas system using Aromatoleum aromaticum EbN1 as a model organism. The cas genes and a minimal CRISPR array of the A. aromaticum EbN1 (type IV CRISPR-Cas system) were transferred into Escherichia coli BL21 AI to uncover the RNA and protein components of this CRISPR-Cas type. Type IV crRNAs were shown to yield unusually short 7 nucleotide 5′-repeat tags and stable 3′ hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR–ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained and the active site of the enzyme was determined. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified the cRNPs’ protein–protein and protein–RNA contact sites. A possible PAM-sequence dependent DNA targeting mechanism of this complex and the involvement of a type IV CRISPR-Cas associated DinG helicase are discussed.
CRISPR-Cas, Prokaryotic Defense Systems
Özcan, Ahsen
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Özcan, Ahsen (2019): Characterization of the type IV CRISPR-Cas system of aromatoleum aromaticum EbN1. Dissertation, LMU München: Faculty of Biology
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Abstract

CRISPR-Cas systems provide prokaryotic adaptive immunity against invading agents, which also stimulated the development of indispensable tools in biological research. CRISPR-Cas systems show remarkable diversity and encompass two classes, six types and 16 subtypes. Among these six computationally classified CRISPR-Cas types, type IV CRISPR-Cas systems remained the only one without experimental data. In this study, we provide the first experimental characterization of a type IV CRISPR-Cas system using Aromatoleum aromaticum EbN1 as a model organism. The cas genes and a minimal CRISPR array of the A. aromaticum EbN1 (type IV CRISPR-Cas system) were transferred into Escherichia coli BL21 AI to uncover the RNA and protein components of this CRISPR-Cas type. Type IV crRNAs were shown to yield unusually short 7 nucleotide 5′-repeat tags and stable 3′ hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR–ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained and the active site of the enzyme was determined. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified the cRNPs’ protein–protein and protein–RNA contact sites. A possible PAM-sequence dependent DNA targeting mechanism of this complex and the involvement of a type IV CRISPR-Cas associated DinG helicase are discussed.