On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires

ABSTRACT Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reve...

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Autores principales:	Sukrit Silas, Kira S. Makarova, Sergey Shmakov, David Páez-Espino, Georg Mohr, Yi Liu, Michelle Davison, Simon Roux, Siddharth R. Krishnamurthy, Becky Xu Hua Fu, Loren L. Hansen, David Wang, Matthew B. Sullivan, Andrew Millard, Martha R. Clokie, Devaki Bhaya, Alan M. Lambowitz, Nikos C. Kyrpides, Eugene V. Koonin, Andrew Z. Fire
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CRISPR RNA spacer acquisition cyanobacteria deep sequencing horizontal gene transfer host-parasite relationship Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a69cf1b12c9e44f9b5265cbf1b3feb45
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spelling	oai:doaj.org-article:a69cf1b12c9e44f9b5265cbf1b3feb452021-11-15T15:51:43ZOn the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires10.1128/mBio.00897-172150-7511https://doaj.org/article/a69cf1b12c9e44f9b5265cbf1b3feb452017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00897-17https://doaj.org/toc/2150-7511ABSTRACT Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.Sukrit SilasKira S. MakarovaSergey ShmakovDavid Páez-EspinoGeorg MohrYi LiuMichelle DavisonSimon RouxSiddharth R. KrishnamurthyBecky Xu Hua FuLoren L. HansenDavid WangMatthew B. SullivanAndrew MillardMartha R. ClokieDevaki BhayaAlan M. LambowitzNikos C. KyrpidesEugene V. KooninAndrew Z. FireAmerican Society for MicrobiologyarticleCRISPRRNA spacer acquisitioncyanobacteriadeep sequencinghorizontal gene transferhost-parasite relationshipMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR RNA spacer acquisition cyanobacteria deep sequencing horizontal gene transfer host-parasite relationship Microbiology QR1-502
spellingShingle	CRISPR RNA spacer acquisition cyanobacteria deep sequencing horizontal gene transfer host-parasite relationship Microbiology QR1-502 Sukrit Silas Kira S. Makarova Sergey Shmakov David Páez-Espino Georg Mohr Yi Liu Michelle Davison Simon Roux Siddharth R. Krishnamurthy Becky Xu Hua Fu Loren L. Hansen David Wang Matthew B. Sullivan Andrew Millard Martha R. Clokie Devaki Bhaya Alan M. Lambowitz Nikos C. Kyrpides Eugene V. Koonin Andrew Z. Fire On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
description	ABSTRACT Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.
format	article
author	Sukrit Silas Kira S. Makarova Sergey Shmakov David Páez-Espino Georg Mohr Yi Liu Michelle Davison Simon Roux Siddharth R. Krishnamurthy Becky Xu Hua Fu Loren L. Hansen David Wang Matthew B. Sullivan Andrew Millard Martha R. Clokie Devaki Bhaya Alan M. Lambowitz Nikos C. Kyrpides Eugene V. Koonin Andrew Z. Fire
author_facet	Sukrit Silas Kira S. Makarova Sergey Shmakov David Páez-Espino Georg Mohr Yi Liu Michelle Davison Simon Roux Siddharth R. Krishnamurthy Becky Xu Hua Fu Loren L. Hansen David Wang Matthew B. Sullivan Andrew Millard Martha R. Clokie Devaki Bhaya Alan M. Lambowitz Nikos C. Kyrpides Eugene V. Koonin Andrew Z. Fire
author_sort	Sukrit Silas
title	On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
title_short	On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
title_full	On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
title_fullStr	On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
title_full_unstemmed	On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires
title_sort	on the origin of reverse transcriptase-using crispr-cas systems and their hyperdiverse, enigmatic spacer repertoires
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/a69cf1b12c9e44f9b5265cbf1b3feb45
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On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires

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