CRISPR interference directs strand specific spacer acquisition.

<h4>Background</h4>CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts o...

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Autores principales:	Daan C Swarts, Cas Mosterd, Mark W J van Passel, Stan J J Brouns
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/af91329f8e294d7c85a725d3c62264d8
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spelling	oai:doaj.org-article:af91329f8e294d7c85a725d3c62264d82021-11-18T07:20:31ZCRISPR interference directs strand specific spacer acquisition.1932-620310.1371/journal.pone.0035888https://doaj.org/article/af91329f8e294d7c85a725d3c62264d82012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22558257/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.<h4>Results</h4>We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner.<h4>Conclusions</h4>The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster--in our case the first acquired spacer--and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.Daan C SwartsCas MosterdMark W J van PasselStan J J BrounsPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 4, p e35888 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daan C Swarts Cas Mosterd Mark W J van Passel Stan J J Brouns CRISPR interference directs strand specific spacer acquisition.
description	<h4>Background</h4>CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.<h4>Results</h4>We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner.<h4>Conclusions</h4>The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster--in our case the first acquired spacer--and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.
format	article
author	Daan C Swarts Cas Mosterd Mark W J van Passel Stan J J Brouns
author_facet	Daan C Swarts Cas Mosterd Mark W J van Passel Stan J J Brouns
author_sort	Daan C Swarts
title	CRISPR interference directs strand specific spacer acquisition.
title_short	CRISPR interference directs strand specific spacer acquisition.
title_full	CRISPR interference directs strand specific spacer acquisition.
title_fullStr	CRISPR interference directs strand specific spacer acquisition.
title_full_unstemmed	CRISPR interference directs strand specific spacer acquisition.
title_sort	crispr interference directs strand specific spacer acquisition.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/af91329f8e294d7c85a725d3c62264d8
work_keys_str_mv	AT daancswarts crisprinterferencedirectsstrandspecificspaceracquisition AT casmosterd crisprinterferencedirectsstrandspecificspaceracquisition AT markwjvanpassel crisprinterferencedirectsstrandspecificspaceracquisition AT stanjjbrouns crisprinterferencedirectsstrandspecificspaceracquisition
_version_	1718423618757591040

CRISPR interference directs strand specific spacer acquisition.

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