Detection of spacer precursors formed in vivo during primed CRISPR adaptation

Shiriaeva, Anna; Savitskaya, Ekaterina; Datsenko, Kirill A.; Vvedenskaya, Irina O.; Fedorova, Iana; Morozova, Natalia; Metlitskaya, Anastasia; Sabantsev, Anton; Nickels, Bryce E.; Severinov, Konstantin; Semenova, Ekaterina

doi:10.1038/s41467-019-12417-w

Cited by 24 publications

(62 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S6). The result is an asymmetric intermediate, which is consistent with recent evidence that in vivo, spacer precursors detected during primed adaptation in both type I-E and type I-F systems share an asymmetrical structure characterized by a blunt non-PAM end and a 3Ј overhang at the PAM end (45).…”

Section: Editors' Pick: Prespacer Processing In E Colisupporting

confidence: 89%

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Ramachandran

Summerville

Learn

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3′ overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3′–5′ exonucleases DnaQ and ExoT can trim long 3′ overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3′–5′ exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

show abstract

Section: Editors' Pick: Prespacer Processing In E Colisupporting

confidence: 89%

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Ramachandran

Summerville

Learn

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Most likely, the CRISPR system was unable to eliminate the prophage from the genome, followed by primed adaptation of additional spacers from locations in the prophage vicinity. Interestingly, as priming is enhanced by CRISPR interference (14,36,37), it is striking that no apparent DNA damage was incurred. For M. elsdenii we found that all STS protospacers are on the same strand with an orientation bias characteristic of primed adaptation (14).…”

Section: Amplified Self-targeting In Prophages Regionsmentioning

confidence: 99%

Prophages are associated with extensive CRISPR-Cas auto-immunity

Nóbrega

Walinga

Dutilh

et al. 2020

Preprint

View full text Add to dashboard Cite

CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS may reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a gene regulatory mechanism. Here we investigated the incidence, distribution, and evasion of STS in over 100,000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of tolerance to STS by other mechanisms such as anti-CRISPR proteins and target mutations. We propose a scenario where it is common and perhaps beneficial to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects. The mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages, and contribute both to viral dissemination and bacterial diversification.

show abstract

“…The presence of consensus PAM sequence (AAG/CTT) increased the acquisition efficiency of prespacers ∼5-fold and ensured specific orientation of integration with the PAM-derived G/C immediately following the first repeat [57]. We developed a method for strand-specific HTS of short DNA fragments generated in vivo that we called FragSeq and that allowed us to detect prespacers in E. coli cells undergoing primed CRISPR adaptation ( Figure 5B) [79]. The method uses protocols that avoid the loss of short fragments during purification of DNA from cell lysates ( phenol-chloroform DNA purification) and size selection of spacer-sized fragments prior to [79] and primer extension [63].…”

Section: Detection Of Spacer Precursors In Vivomentioning

confidence: 99%

“…We developed a method for strand-specific HTS of short DNA fragments generated in vivo that we called FragSeq and that allowed us to detect prespacers in E. coli cells undergoing primed CRISPR adaptation ( Figure 5B) [79]. The method uses protocols that avoid the loss of short fragments during purification of DNA from cell lysates ( phenol-chloroform DNA purification) and size selection of spacer-sized fragments prior to [79] and primer extension [63]. A prespacer (blue and red rectangles) in DNA (blue and red lines) is bound by the Cas1-Cas2 complex (light green).…”

Section: Detection Of Spacer Precursors In Vivomentioning

confidence: 99%

“…In our work, we used FragSeq to analyze prespacers formed during primed adaptation [79]. The hallmark of primed adaptation by the type I-E CRISPR-Cas system is the acquisition of spacers with the sequence of the nontranscribed strand originating from the nontarget strand of the DNA degraded by CRISPR interference machinery [30,31].…”

Section: Detection Of Spacer Precursors In Vivomentioning

confidence: 99%

See 1 more Smart Citation

Detection of CRISPR adaptation

Shiriaeva

Fedorov

Vyhovskyi

et al. 2020

Biochemical Society Transactions

Self Cite

View full text Add to dashboard Cite

Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

show abstract

Detection of spacer precursors formed in vivo during primed CRISPR adaptation

Cited by 24 publications

References 38 publications

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Prophages are associated with extensive CRISPR-Cas auto-immunity

Detection of CRISPR adaptation

Contact Info

Product

Resources

About