Biology subjects

Jakob, L.

Publications and source records attributed to Jakob, L..

3 recordsLinked to original sources

bioRxiv2022

Antibiotic-induced dysbiosis does not potentiate the effect of lipopolysaccharide insult in male Wistar rats

The present study investigated whether neonatal exposure to the proinflammatory endotoxin lipopolysaccharide (LPS) followed by antibiotic (ATB)-induced dysbiosis in early adulthood could induce schizophrenia-like behavioral changes in adult male rats. The combination of these two stressors resulted in decreased weight gain, but no significant behavioral abnormalities were observed. LPS treatment resulted in adult rats hypoactivity and induced anxiety-like behavior in the social recognition paradigm, but these behavioral changes were not exacerbated by ATB-induced gut dysbiosis. ATB treatment seriously disrupted the gut bacterial community, but dysbiosis did not affect locomotor activity, social recognition, and acoustic reactivity in adult rats. Fecal bacterial community analyzes showed no differences between the LPS challenge exposed/unexposed rats, while the effect of ATB administration was decisive regardless of prior LPS exposure. ATB treatment resulted in significantly decreased bacterial diversity, suppression of Clostridiales and Bacteroidales, and increases in Lactobacillales, Enterobacteriales, and Burkholderiales. The persistent effect of LPS on some aspects of behavior suggests a long-term effect of early toxin exposure that was not observed in ATB-treated animals. However, an anti-inflammatory protective effect of ATB cannot be assumed because of the increased abundance of pro-inflammatory, potentially pathogenic bacteria (Proteus, Suttrella) and the elimination of the bacterial families Ruminococcaceae and Lachnospiraceae, which are generally considered beneficial for gut health.

neuroscience↗

bioRxiv2021

Probing the stability of the SpCas9-DNA complex after cleavage

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

biophysics↗

bioRxiv2021

Decoupling the bridge helix of Cas12a results in a reduced trimming activity and impaired conformational transitions

The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by a central structural element, the so-called bridge helix (BH). We generated BH mutants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. Single-molecule FRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH mutants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH mutants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.

biochemistry↗