Biology subjects

Patinios, C.

Publications and source records attributed to Patinios, C..

4 recordsLinked to original sources

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↗

bioRxiv2023

Type III-B CRISPR-Cas signaling-based cascade of proteolytic cleavages

Type III CRISPR-Cas systems provide a sequence-specific adaptive immune response that protects prokaryotic hosts against viruses and other foreign genetic invaders. These crRNA-guided Cas effector complexes bind and cleave complementary RNA targets. Specific target binding stimulates the Cas10 subunit to generate cyclic oligoadenylate (cOA) signaling molecules, that in turn allosterically activate proteins carrying cognate sensory domains: CARF or SAVED. Here, we characterize an elaborate set of genes associated with the type III-B CRISPR-Cas system from Haliangium ochraceum, which includes a signal transduction module of a CBASS defense system with two caspase-like proteases, SAVED-CHAT and PCaspase (Prokaryotic Caspase). We show that binding of a 3-nucleotide cOA (cA3) to the SAVED domain of SAVED-CHAT induces its oligomerization into long filaments that activate the proteolytic activity of the CHAT domain. Surprisingly, we find that activated SAVED-CHAT specifically cleaves and activates the second protease, PCaspase. In turn, activated PCaspase cleaves a multitude of other proteins, including a putative sigma factor and a PCaspase-inhibitor. We expressed the type III-B system and its associated genes in E. coli and observed a strong abortive phenotype when offering a complementary target RNA, but only in the presence of both SAVED-CHAT and PCaspase. Together, our findings show an intriguing cascade of proteolytic activities (conceptually similar to eukaryotic caspases) in this bacterial immune system that reveals yet another strategy to effectively defend against mobile genetic elements.

biochemistry↗

bioRxiv2022

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of several CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the strict xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (Spo0A, Upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the Spo0A and Upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

microbiology↗

bioRxiv2021

SIBR-Cas enables host-independent and universal CRISPR genome engineering in bacteria

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and universal genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for universal and inducible control over CRISPR-Cas counterselection. This control delays CRISPR-Cas counterselection, granting more time for the editing event (e.g., by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three bacteria with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a universal gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

bioengineering↗