CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR-Cas system

Nucleic Acids Research
Jenny G KimMichael P Terns

Abstract

CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to ...Continue Reading

References

Apr 16, 2002·Molecular Microbiology·Ruud JansenLeo M Schouls
Jun 3, 2004·Genome Research·Gavin E CrooksSteven E Brenner
Mar 29, 2005·Journal of Molecular Evolution·Francisco J M MojicaElena Soria
Mar 24, 2007·Science·Rodolphe BarrangouPhilippe Horvath
Dec 11, 2007·Journal of Bacteriology·Philippe HorvathRodolphe Barrangou
Dec 11, 2007·Journal of Bacteriology·Hélène DeveauSylvain Moineau
Aug 16, 2008·Science·Stan J J BrounsJohn van der Oost
Oct 31, 2008·RNA·Caryn HaleMichael P Terns
Jan 15, 2009·Genes & Development·Jason CarteMichael P Terns
Mar 6, 2009·Genome Biology·Ben LangmeadSteven L Salzberg
Jan 30, 2010·Bioinformatics·Aaron R Quinlan, Ira M Hall
Nov 5, 2010·Nature·Josiane E GarneauSylvain Moineau
Apr 2, 2011·Nature·Elitza DeltchevaEmmanuelle Charpentier
Aug 23, 2012·PloS One·Alfonso H MagadánSylvain Moineau
May 6, 2014·Nature Structural & Molecular Biology·James K NuñezJennifer A Doudna
Jun 10, 2014·Nature Reviews. Microbiology·John van der OostBlake Wiedenheft
Nov 5, 2014·Genes & Development·Caryn R HaleMichael P Terns
Feb 19, 2015·Genes & Development·Yunzhou WeiMichael P Terns
Feb 25, 2015·Nature·James K NuñezJennifer A Doudna
Feb 25, 2015·Nature·Robert HelerLuciano A Marraffini
May 23, 2015·FEMS Microbiology Reviews·Emmanuelle CharpentierMalcolm F White
Sep 29, 2015·Nature Reviews. Microbiology·Kira S MakarovaEugene V Koonin
Oct 4, 2015·Nature·Luciano A Marraffini
Oct 28, 2015·Nature·James K NuñezJennifer A Doudna
Nov 19, 2015·Nucleic Acids Research·Ivana Ivančić-BaćeEdward L Bolt
Jan 12, 2016·Nature Reviews. Microbiology·Gil Amitai, Rotem Sorek
Mar 8, 2016·Molecular Cell·Samuel H SternbergUdi Qimron
May 24, 2016·Molecular Cell·James K NuñezJennifer A Doudna
Aug 23, 2016·Molecular Cell·Tim KünneStan J J Brouns
Sep 7, 2016·Nature Structural & Molecular Biology·Addison V Wright, Jennifer A Doudna
Apr 8, 2017·Science·Simon A JacksonStan J J Brouns
Jun 13, 2017·Current Opinion in Microbiology·Eugene V KooninFeng Zhang

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Citations

Jun 10, 2020·The Journal of Biological Chemistry·Mason J Van OrdenRakhi Rajan
Apr 20, 2021·Frontiers in Microbiology·Sandra C Garrett
Jun 19, 2021·Nature Communications·Katerina ZabradyAidan J Doherty
Jun 27, 2021·Current Biology : CB·Andrew Santiago-FrangosBlake Wiedenheft
Dec 12, 2021·Nucleic Acids Research·Xinfu ZhangMichael P Terns

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Datasets Mentioned

BETA
PRJNA548779

Methods Mentioned

BETA
PCR
column chromatography
electrophoresis
X-ray

Software Mentioned

bedtools
bowtie

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