Biology subjects

Songailiene, I.

Publications and source records attributed to Songailiene, I..

4 recordsLinked to original sources

bioRxiv2023

Astonishing diversity and multifaceted biological connections of Type IV restriction-modification systems

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote CoCoNuTs (coiled-coil nuclease tandems) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with 3 distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

microbiology↗

bioRxiv2023

The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex

The discovery1,2 and the pioneering applications3 of CRISPR-Cas effector complexes have provided powerful gene-editing tools. The effector complexes are guided to the targeted genomic locus by the complementarity of their CRISPR RNA (crRNA)4,5. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base-pairing between crRNA and the DNA target strand resulting in the formation of an R-loop structure5,6. Full R-loop formation is the prerequisite for the subsequent DNA cleavage. While the CRISPR-Cas technology is easy to use, efficient and highly versatile, therapeutic applications are hampered by the off-target effects due to the recognition of unintended sequences with multiple mismatches7. This process is still poorly understood on a mechanistic level8,9. Particularly, the lack of insight into the energetics and dynamics of the R-loop formation hinders a direct modelling of the R-loop formation for off-target prediction. Here we set up ultrafast DNA unwinding experiments based on plasmonic DNA nanorotors to follow the R-loop formation by the Cascade effector complex in real time, close to base pair resolution. We directly resolve a weak global downhill bias of the energy landscape of the forming R-loop followed by a steep uphill bias for the final base pairs. We furthermore show a modulation of the landscape by base flips and mismatches. These data provide that Cascade-mediated R-loop formation occurs on short time scales in single base pair steps of sub-millisecond duration, but on longer time scales in six-base pair intermediate steps in agreement with the structural periodicity of the crRNA-DNA hybrid. We expect that the knowledge about the energy landscapes of R-loop formation of CRISPR-Cas effector complexes will pave the way for a detailed understanding and prediction of off-target recognition10.

biophysics↗

bioRxiv2022

Dynamic interplay between target search and recognition for the Cascade surveillance complex of type I-E CRISPR-Cas systems

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors first have to interrogate the entire genome for the presence of a matching sequence. Matching is achieved by base-pairing between the crRNA of the complexes and the DNA target strand such that an R-loop is formed. R-loop formation starts at a specific PAM motif and progresses reversibly in single base-pair steps until mismatches stop further progression or until the full target is recognized and destroyed. The reversible nature of this process entails that even a fully matching target should only become recognized with a low probability per target encounter. The details of this process, which directly affect the effectiveness of the target search, remain unresolved. Here we dissect the target search process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the low target recognition probabilities and show that they increase with increasing negative supercoiling. Furthermore, we demonstrate that Cascade uses a combination of three-dimensional and limited one-dimensional diffusion along the DNA contour for its target search. The latter allows for rapidly scanning the PAM sequences in a given region and, importantly, significantly increasing the overall efficiency of the target search by repeatedly revisiting the sites. Overall we show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

biophysics↗

bioRxiv2022

A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex

CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.

biophysics↗