A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Workman, Rachael E.; Pammi, Teja; Nguyen, Binh T.; Graeff, Leonardo W.; Smith, Erika; Sebald, Suzanne M.; Stoltzfus, Marie J.; Euler, Chad W.; Modell, Joshua W.

doi:10.1016/j.cell.2020.12.017

Cited by 50 publications

(60 citation statements)

References 84 publications

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“…Starting from the characterization of a native CRISPR-Cas9 system in the bacterial pathogen C. jejuni , we discovered that cellular transcripts can be the source of noncanonical crRNAs through hybridization with the tracrRNA. This discovery adds ncrRNAs to the list of RNA guides found in nature, including crRNAs, scaRNAs that similarly pair with the tracrRNA anti-repeat, and “natural” sgRNAs formed through upstream transcription of the tracrRNA ( 30 , 31 ). These prior examples are all encoded within CRISPR-Cas loci.…”

Section: Discussionmentioning

confidence: 99%

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Jiao

Sharma

Dugar

et al. 2021

Science

101

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CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.

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Section: Discussionmentioning

confidence: 99%

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Jiao

Sharma

Dugar

et al. 2021

Science

101

View full text Add to dashboard Cite

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“…6A). Also the annotated tracrRNA of the type II-A CRISPR-Cas system has recently been shown to cause mitigation of autoimmunity but also at the cost of reduced bacteriophage immunity 41 .…”

Section: Highly Variable Phage Resistance Mechanisms In Isolated Genomesmentioning

confidence: 99%

Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

Somerville

Berthoud

Schmidt

et al. 2021

Preprint

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Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

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“…A recent study discovered a new regulatory mechanism involving a long-form tracrRNA, capable of autoregulating CRISPR-Cas expression and especially cas gene transcription, to avoid autoimmunity. 43 The tracr-L binds to cas promoter, which dampens transcription. While this regulatory mechanism may occur in the ST23 strain, the tracr-L and its target are deleted in the ST17 strain.…”

Section: Streptococcus Agalactiae St17 Crispr-cas Systemmentioning

confidence: 99%

Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains

et al. 2021

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Nearly all strains of Streptococcus agalactiae, the leading cause of invasive infections in neonates, encode a type II-A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Interestingly, S. agalactiae strains belonging to the hypervirulent Sequence Type 17 (ST17) contain significantly fewer spacers in their CRISPR locus than other lineages, which could be the result of a less functional CRISPR-Cas system. Here, we revealed one large deletion in the ST17 cas promoter region and we evaluated its impact on the transcription of cas genes as well as the functionalities of the CRISPR-Cas system. We demonstrated that Cas9 interference is functional and that the CRISPR-Cas system of ST17 strains can still acquire new spacers, despite the absence of a regular cas promoter. We demonstrated that a promoter sequence upstream of srn036, a small RNA partially overlapping the antisense tracrRNA, is responsible for the ST17 CRISPR-Cas adaptation and interference activities.

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A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Cited by 50 publications

References 84 publications

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains

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