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DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system
BACKGROUND: CRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism invo...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296934/ https://www.ncbi.nlm.nih.gov/pubmed/32539804 http://dx.doi.org/10.1186/s12915-020-00799-z |
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author | Tuminauskaite, Donata Norkunaite, Danguole Fiodorovaite, Marija Tumas, Sarunas Songailiene, Inga Tamulaitiene, Giedre Sinkunas, Tomas |
author_facet | Tuminauskaite, Donata Norkunaite, Danguole Fiodorovaite, Marija Tumas, Sarunas Songailiene, Inga Tamulaitiene, Giedre Sinkunas, Tomas |
author_sort | Tuminauskaite, Donata |
collection | PubMed |
description | BACKGROUND: CRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems. RESULTS: Here we examined the mechanism of DNA interference provided by the type I-F1 system from Aggregatibacter actinomycetemcomitans D7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32 nt but also with shorter or longer (14–176 nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at −1 or −2,−1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage. CONCLUSIONS: We provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop. |
format | Online Article Text |
id | pubmed-7296934 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-72969342020-06-16 DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system Tuminauskaite, Donata Norkunaite, Danguole Fiodorovaite, Marija Tumas, Sarunas Songailiene, Inga Tamulaitiene, Giedre Sinkunas, Tomas BMC Biol Research Article BACKGROUND: CRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems. RESULTS: Here we examined the mechanism of DNA interference provided by the type I-F1 system from Aggregatibacter actinomycetemcomitans D7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32 nt but also with shorter or longer (14–176 nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at −1 or −2,−1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage. CONCLUSIONS: We provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop. BioMed Central 2020-06-15 /pmc/articles/PMC7296934/ /pubmed/32539804 http://dx.doi.org/10.1186/s12915-020-00799-z Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Article Tuminauskaite, Donata Norkunaite, Danguole Fiodorovaite, Marija Tumas, Sarunas Songailiene, Inga Tamulaitiene, Giedre Sinkunas, Tomas DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system |
title | DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system |
title_full | DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system |
title_fullStr | DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system |
title_full_unstemmed | DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system |
title_short | DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system |
title_sort | dna interference is controlled by r-loop length in a type i-f1 crispr-cas system |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296934/ https://www.ncbi.nlm.nih.gov/pubmed/32539804 http://dx.doi.org/10.1186/s12915-020-00799-z |
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