Biology subjects

Migur, A.

Publications and source records attributed to Migur, A..

5 recordsLinked to original sources

bioRxiv2026

Phage-encoded single-guide RNAs subvert CRISPR-Cas9 immunity

Central to CRISPR technologies is the single-guide RNA (sgRNA), an engineered fusion of a processed CRISPR RNA and tracrRNA that directs Cas9 and many Cas12 nucleases to bind and cleave target DNA1-4. Here, we report the discovery of similarly compact viral sgRNAs (vsgRNAs) encoded by bacteriophages that counteract bacterial CRISPR-Cas9 immunity. vsgRNAs inhibit Cas9 function via two complementary routes: by sequestering the Cas9 apoenzyme, and by re-directing the Cas9 nuclease to transcriptionally silence its own promoter. vsgRNAs also can cooperate with co-encoded anti-CRISPR proteins (Acrs), including AcrIIA25.1 that blocks DNA binding by Cas9 complexed with a standard sgRNA but not with the vsgRNA. We predict that phages evolved vsgRNAs by co-opting and repurposing host-encoded long-form tracrRNAs (tracr-L) responsible for Cas9 auto-repression and a countermeasure to Acrs5,6. Our search also uncovered Cas9-regulating small CRISPR-associated RNAs (scaRNAs), which we predict were inserted upstream of tracrRNAs to form tracr-L but were also co-opted by phages as viral scaRNAs to suppress Cas9 immunity. Finally, we found that vsgRNAs can enable genome editing in mammalian cells, offering a natural guide RNA template for CRISPR technologies. Overall, these findings reveal that bacteriophages devised their own compact sgRNAs tailored to subvert Cas9 immunity, long preceding their rational design to program RNA-guided nucleases2.

microbiology↗

bioRxiv2024

Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

synthetic biology↗

bioRxiv2024

AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack

Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly-bound crRNA does not undergo processing and exhibits altered target RNA binding that fails to activate collateral RNA cleavage. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryo-EM structures revealed that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain in an untethered state. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense. HighlightsO_LIAcrVIB1 binds Cas13b in the absence of a crRNA C_LIO_LIThe bound AcrVIB1 promotes crRNA binding to Cas13b C_LIO_LIThe crRNA binds Cas13b unproductively and is accessible to RNases C_LIO_LIAcrVIB1 binds the Helical-2 domain of Cas13b, preventing it from securing the crRNA C_LI

biochemistry↗

bioRxiv2023

Control of a type III-Dv CRISPR-Cas system by the transcription factor RpaB and interaction of its transcribed leader with the DEAD-box RNA helicase CrhR

CRISPR-Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR-Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacterium Synechocystis sp. PCC 6803 encodes three different CRISPR-Cas systems. The expression of one of these, a type III-Dv system, responds to changes in environmental conditions, such as nitrogen starvation or varying light intensities. Here, we found that the promoter of the six-gene cas operon for the type III-Dv system is controlled by the light-and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif located 53 to 70 nt upstream of the transcription start site, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, we found that the 125 nt leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled to mass spectrometry analysis revealed six residues involved in the CrhR-RNA interaction. Of these, L103, F104, H225, and C371 were predicted to be on the surface of a dimeric CrhR model, while C184 was not on the surface, and P443 could not be assigned to a structural element. These results showed that the expression of the CRISPR-Cas system is linked to the redox status of the photosynthetic cyanobacterial cell at two different levels. While RpaB affects transcription, CrhR interacts with the leader transcript posttranscription. These results highlight the complex interplay between a CRISPR-Cas system and its host cell.

microbiology↗

bioRxiv2021

The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the {Delta}crhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets. HighlightThe cyanobacterial DEAD-box RNA helicase CrhR binds mainly photosynthesis-associated and redox-controlled transcripts connecting its regulation, localization and phenotypes of mutants for the first time with a set of potential RNA targets.

plant biology↗