Feel the burn: a collection of stories on hot’n’sharp DNA engineering

The global demand on chemicals and fuels is exponentially increasing. At the same time, the excessive exploitation of fossil-based resources for the coverage of this demand has a high environmental impact, motivating the production of green chemicals and biofuels from renewable resources. Nowadays, the microbial production of green chemicals and fuels gains increasing attention, especially due to the ease in the construction of metabolically engineered microorganisms with high production capacities. This ease was achieved via the development of efficient genome engineering tools. This thesis describes the development of novel genetic tools for mesophilic and thermophilic bacteria, for metabolic engineering purposes. More specifically, here is reported: 1) the development of the first CRISPR-SpCas9-based genome engineering tools for the mesophilic bacteria Rhodobacter sphaeroides and Pseudomonas putida, as well as for the moderate thermophilic bacterium Bacillus smithii, 2) the in vitro characterization of one of the first reported thermotolerant Cas9 homologs, denoted as ThermoCas9, as well the development of the first CRISPR-ThermoCas9-based genome engineering tools for strictly thermophilic bacteria, and 3) the use of the developed tools for the metabolic exploration and exploitation of the moderate thermophilic bacterium B. smithii, towards the characterization of its acetate production pathway and the enhancement of its dicarboxylic acids productivity.</p

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range

Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering : advances and prospects

High engineering efficiencies are required for industrial strain development. Due to its user-friendliness and its stringency, CRISPR-Cas-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host Escherichia coli and non-model organisms like Clostridia, Bacilli, Streptomycetes and cyanobacteria, opening new possibilities to use these organisms as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of organisms in which it can be used to create novel production hosts. This review analyses the current status of prokaryotic metabolic engineering towards the production of biotechnologically relevant products, based on the exploitation of different CRISPR-related DNA/RNA endonuclease variants

Pharmacoeconomic analysis of paliperidone palmitate for treating schizophrenia in Greece

BACKGROUND: Patients having chronic schizophrenia with frequent relapses and hospitalizations represent a great challenge, both clinically and financially. Risperidone long-acting injection (RIS-LAI) has been the main LAI atypical antipsychotic treatment in Greece. Paliperidone palmitate (PP-LAI) has recently been approved. It is dosed monthly, as opposed to biweekly for RIS-LAI, but such advantages have not yet been analysed in terms of economic evaluation. PURPOSE: To compare costs and outcomes of PP-LAI versus RIS-LAI in Greece. METHODS: A cost-utility analysis was performed using a previously validated decision tree to model clinical pathways and costs over 1 year for stable patients started on either medication. Rates were taken from the literature. A local expert panel provided feedback on treatment patterns. All direct costs incurred by the national healthcare system were obtained from the literature and standard price lists; all were inflated to 2011 costs. Patient outcomes analyzed included average days with stable disease, numbers of hospitalizations, emergency room visits, and quality-adjusted life-years (QALYs). RESULTS: The total annual healthcare cost with PP-LAI was €3529; patients experienced 325 days in remission and 0.840 QALY; 28% were hospitalized and 15% received emergency room treatment. With RIS-LAI, the cost was €3695, patients experienced 318.6 days in remission and 0.815 QALY; 33% were hospitalized and 17% received emergency room treatment. Thus, PP-LAI dominated RIS-LAI. Results were generally robust in sensitivity analyses with PP-LAI dominating in 74.6% of simulations. Results were sensitive to the price of PP-LAI. CONCLUSIONS: PP-LAI appears to be a cost-effective option for treating chronic schizophrenia in Greece compared with RIS-LAI since it results in savings to the health care system along with better patient outcomes

First structural insights into CRISPR-Cas-guided DNA transposition

The transposition mechanism of the Vibrio cholerae Tn6677 transposon (VcTn6677) is based on the unique synergy between a classical transposition machinery (TnsABC-TniQ complex) and a nuclease-deficient type I-F CRISPR-associated complex for antiviral defense (VcCascade). Four independent studies, three of which appeared in Cell Research, recently reported structures of the VcCascade, VcCascade-TniQ, and/or VcCascade-TniQ-dsDNA complexes as well as a TniQ dimer complex, providing the first insights into the assembly and DNA targeting mechanism of a CRISPR-associated transposase complex

Base editing tools

Thermostable, type II-C Cas9 variants of Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) use 23 nt spacers to edit sites adjacent to an N4CVAA/N4CCCA or N4CRAA PAM, respectively. These Cas9 variants are linked to a cytidine deaminase enzyme from the sea lamprey Petromyzon marinus (PmCDA1) to provide: (a) dThermoTarget-AID, (b) dGeoTarget- AID, (c) AcrThermoTarget-AID, and (d) AcrGeoTarget-AID. Compared to known base editors, these systems exhibit much larger base editing windows: (a) from -5 to -27 positions relative to the PAM (23 bp); (b) from -5 to -24 positions relative to the PAM (20 bp); (c) from -5 to -27 positions relative to the PAM (23 pb); (d) from -3 to - 28 positions relative to the PAM (26 bp). The first two editors employ the catalytically inactive ThermoCas9 or GeoCas9, respectively, while the last two editors co-express a small anti-CRISPR protein from Neisseria meningitidis (AcrIIC1Nme) with active ThermoCas9 or GeoCas9, respectively. Methods of gene editing are described