The spacer size of I-B CRISPR is modulated by the terminal sequence of the protospacer

Li, Ming; Gong, Luyao; Zhao, Dahe; Zhou, Jian; Xiang, Hua

doi:10.1093/nar/gkx229

Cited by 20 publications

(27 citation statements)

References 40 publications

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“…3C). This downstream GAA signal is consistent with a phenomenon known as "flipping"-where the prespacer substrate next to a TTC PAM is integrated in an opposite direction into the CRISPR array so that the reverse complementary strand is used as a spacer (Shmakov et al 2014;Staals et al 2016;Li et al 2017). Indeed, we identified ∼1% spacers derived from potential flips with an original TTC PAM, 0.3%-0.4% spacers from a combination of flips and +1 nt slips, and even rarer still-combinations of flips and other types of slips (Fig.…”

Section: '-Caagaccgaaggcggacggcagctccggctgttcctggaacccaagtgggaggccgtsupporting

confidence: 67%

“…With respect to PAM preference, in addition to the canonical TTC PAM (∼90% of all acquired spacers), we identified an alternative TTT PAM (2%-4%) and extensive slipping and flipping events (6%-8%). With respect to spacer size selection, we observed a flexible choice more similar to the type I-B system (Li et al 2017) than the slightly more stringent type I-E (Savitskaya et al 2013;Fineran et al 2014) and type I-F (Richter et al 2014;Staals et al 2016) systems. A recent study on the length variation of acquired spacers in the H. hispanica type I-B system showed that the nucleotide specificity at both ends of the PAM-protospacer sequence could influence the molecular ruler in the adaptation machinery (Li et al 2017).…”

Section: Discussionmentioning

confidence: 83%

“…S1A). As these less frequent PAMs share a 2 nt identity with TTC, which could derive from slipping events where the real PAM is still a TTC located nearby (Shmakov et al 2014;Staals et al 2016;Li et al 2017), we first suspected that the TTT and TCN PAMs might be due to −1 nt slips (upstream) and +1 nt slips (downstream), respectively ( Fig. 3A).…”

Section: Analysis Of Acquired Spacers Reveals An Alternative Pam and mentioning

confidence: 99%

“…Primed spacer acquisition in the E. coli type I-E system showed a clear preference (>90%) from the untargeted strand and either an extensively long positional gradient on large bacteriophages or a lack of positional gradient on small-sized priming plasmids (Datsenko et al 2012;Savitskaya et al 2013;Fineran et al 2014;Strotskaya et al 2017). In the Haloarcula hispanica type I-B system, ∼70% of new spacers were derived from the untargeted strand and a moderate preference was seen for the priming-proximal region (Li et al 2014b(Li et al , 2017. In the Pectobacterium atrosepticum type I-F system, ∼65% of spacers were acquired from the targeted strand with a clear gradient centered at the priming site (Richter et al 2014;Staals et al 2016).…”

Section: Introductionmentioning

confidence: 99%

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Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss

Rao

Chin

Ensminger

2017

RNA

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CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3'-5' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

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Section: '-Caagaccgaaggcggacggcagctccggctgttcctggaacccaagtgggaggccgtsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 83%

Section: Analysis Of Acquired Spacers Reveals An Alternative Pam and mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss

Rao

Chin

Ensminger

2017

RNA

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“…Genomes carrying type II and V CRISPR systems commonly have non-consensus PAM sequences of the STS protospacer which may help avoid auto-immunity. Whether this occurs by incorrect acquisition of the spacer (43,44), or mutation of the PAM when it is already integrated, is unknown. Although found at low frequency, Acrs were also present significantly (36-fold) more often in STS-containing genomes than STS-free genomes.…”

Section: Discussionmentioning

confidence: 99%

Prophages are associated with extensive CRISPR-Cas auto-immunity

Nóbrega

Walinga

Dutilh

et al. 2020

Preprint

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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.

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Reprogramming the endogenous type I CRISPR‐Cas system for simultaneous gene regulation and editing in Haloarcula hispanica

Gong

et al. 2022

mLife

Self Cite

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The type I system is the most widely distributed CRISPR‐Cas system identified so far. Recently, we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA‐resembling RNA in halophilic archaea. Here, we conducted a comprehensive study of the impact of redesigned crRNAs with different spacer lengths on gene regulation with the native type I‐B CRISPR system in Haloarcula hispanica. When the spacer targeting the chromosomal gene was shortened from 36 to 28 bp, transformation efficiencies of the spacer‐encoding plasmids were improved by over three orders of magnitude, indicating a significant loss of interference. However, by conducting whole‐genome sequencing and measuring the growth curves of the hosts, we still detected DNA cleavage and its influence on cell growth. Intriguingly, when the spacer was shortened to 24 bp, the transcription of the target gene was downregulated to 10.80%, while both interference and primed adaptation disappeared. By modifying the lengths of the spacers, the expression of the target gene could be suppressed to varying degrees. Significantly, by designing crRNAs with different spacer lengths and targeting different genes, we achieved simultaneous gene editing (cdc6E) and gene regulation (crtB) for the first time with the endogenous type I CRISPR‐Cas system.

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The spacer size of I-B CRISPR is modulated by the terminal sequence of the protospacer

Cited by 20 publications

References 40 publications

Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss

Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss

Prophages are associated with extensive CRISPR-Cas auto-immunity

Reprogramming the endogenous type I CRISPR‐Cas system for simultaneous gene regulation and editing in Haloarcula hispanica

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