Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia.

IMPORTANCE: Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

Refined control of CRISPR-Cas9 gene editing in Clostridium sporogenes: the creation of recombinant strains for therapeutic applications.

Despite considerable clinical success, the potential of cancer immunotherapy is restricted by a lack of tumour-targeting strategies. Treatment requires systemic delivery of cytokines or antibodies at high levels to achieve clinically effective doses at malignant sites. This is exacerbated by poor penetration of tumour tissue by therapeutic antibodies. High-grade immune-related adverse events (irAEs) occur in a significant number of patients (5-15%, cancer- and therapeutic-dependent) that can lead to lifelong issues and can exclude from treatment patients with pre-existing autoimmune diseases. Tumour-homing bacteria, genetically engineered to produce therapeutics, is one of the approaches that seeks to mitigate these drawbacks. The ability of Clostridium sporogenes to form spores that are unable to germinate in the presence of oxygen (typical of healthy tissue) offers a unique advantage over other vectors. However, the limited utility of existing gene editing tools hinders the development of therapeutic strains. To overcome the limitations of previous systems, expression of the Cas9 protein and the gRNA was controlled using tetracycline inducible promoters. Furthermore, the components of the system were divided across two plasmids, improving the efficiency of cloning and conjugation. Genome integrated therapeutic genes were assayed biochemically and in cell-based functional assays. The potency of these strains was further improved through rationally-conceived gene knock-outs. The new system was validated by demonstrating the efficient addition and deletion of large sequences from the genome. This included the creation of recombinant strains expressing two pro-inflammatory cytokines, interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF), and a pro-drug converting enzyme (PCE). A comparative, temporal in vitro analysis of the integrant strains and their plasmid-based equivalents revealed a substantial reduction of cytokine activity in chromosome-based constructs. To compensate for this loss, a 7.6 kb operon of proteolytic genes was deleted from the genome. The resultant knock-out strains showed an 8- to 10-fold increase in cytokine activity compared to parental strains.

Streamlined assembly of cloning and genome editing vectors for genus Clostridium.

Reported herein is a new set of vectors designed to streamline molecular cloning and genome editing by exploiting modern cloning methods. The new vectors build on the existing pMTL8000 vectors that have been a staple of Clostridium research for more than a decade. The introduction of two pairs of type IIS restriction sites flanking an insulated multiple cloning site in both a cloning vector and a CRISPR-Cas9 gene editing vector enables plasmid construction in a "one-pot" reaction, avoiding the more laborious steps of conventional cloning. A synthetic lacZα expression cassette introduced between the cloning sites enables visual detection of background colonies. In addition, distinct selection markers on each vector permit selection of the desired clones according to antibiotic resistance. An example of strain development using the new vectors is demonstrated.

Heterologous Gene Regulation in Clostridia: Rationally Designed Gene Regulation for Industrial and Medical Applications.

Several species from the Clostridium genus show promise as industrial solvent producers and cancer therapeutic delivery vehicles. Previous development of shuttle plasmids and genome editing tools has aided the study of these species and enabled their exploitation in industrial and medical applications. Nevertheless, the precise control of gene expression is still hindered by the limited range of characterized promoters. To address this, libraries of promoters (native and synthetic), 5' UTRs, and alternative start codons were constructed. These constructs were tested in Escherichia coli K-12, Clostridium sporogenes NCIMB 10696, and Clostridium butyricum DSM 10702, using ß-glucuronidase (gusA) as a gene reporter. Promoter activity was corroborated using a second gene reporter, nitroreductase (nmeNTR) from Neisseria meningitides. A strong correlation was observed between the two reporters. In C. sporogenes and C. butyricum, respectively, changes in GusA activity between the weakest and strongest expressing levels were 129-fold and 78-fold. Similar results were obtained with the nmeNTR. Using the GusA reporter, translation initiation from six alternative (non-AUG) start codons was measured in E. coli, C. sporogenes, and C. butyricum. Clearly, species-specific differences between clostridia and E. coli in translation initiation were observed, and the performance of the start codons was influenced by the upstream 5' UTR sequence. These results highlight a new opportunity for gene control in recombinant clostridia. To demonstrate the value of these results, expression of the sacB gene from Bacillus subtilis was optimized for use as a novel negative selection marker in C. butyricum. In summary, these results indicate improvements in the understanding of heterologous gene regulation in Clostridium species and E. coli cloning strains. This new knowledge can be utilized for rationally designed gene regulation in Clostridium-mediated industrial and medical applications, as well as fundamental research into the biology of Clostridium species.

Relation between hearing abilities and preferred playback settings for speech perception in complex listening conditions.

OBJECTIVE: This study investigated if individual preferences with respect to the trade-off between a good signal-to-noise ratio and a distortion-free speech target were stable across different masking conditions and if simple adjustment methods could be used to identify subjects as either "noise haters" or "distortions haters". DESIGN: In each masking condition, subjects could adjust the target speech level according to their preferences by employing (i) linear gain or gain at the cost of (ii) clipping distortions or (iii) compression distortions. The comparison of these processing conditions allowed investigating the preferred trade-off between distortions and noise disturbance. STUDY SAMPLE: Thirty subjects differing widely in hearing status (normal-hearing to moderately impaired) and age (23-85 years). RESULTS: High test-retest stability of individual preferences was found for all modification schemes. The preference adjustments suggested that subjects could be consistently categorised along a scale from "noise haters" to "distortion haters", and this preference trait remained stable through all maskers, spatial conditions, and types of distortions. CONCLUSIONS: Employing quick self-adjustment to collect listening preferences in complex listening conditions revealed a stable preference trait along the "noise vs. distortions" tolerance dimension. This could potentially help in fitting modern hearing aid algorithms to the individual user.

Use of an optimised enzyme/prodrug combination for Clostridia directed enzyme prodrug therapy induces a significant growth delay in necrotic tumours.

Necrosis is a typical histological feature of solid tumours that provides a selective environment for growth of the non-pathogenic anaerobic bacterium Clostridium sporogenes. Modest anti-tumour activity as a single agent encouraged the use of C. sporogenes as a vector to express therapeutic genes selectively in tumour tissue, a concept termed Clostridium Directed Enzyme Prodrug Therapy (CDEPT). Here, we examine the ability of a recently identified Neisseria meningitidis type I nitroreductase (NmeNTR) to metabolise the prodrug PR-104A in an in vivo model of CDEPT. Human HCT116 colon cancer cells stably over-expressing NmeNTR demonstrated significant sensitivity to PR-104A, the imaging agent EF5, and several nitro(hetero)cyclic anti-infective compounds. Chemical induction of necrosis in human H1299 xenografts by the vascular disrupting agent vadimezan promoted colonisation by NmeNTR-expressing C. sporogenes, and efficacy studies demonstrated moderate but significant anti-tumour activity of spores when compared to untreated controls. Inclusion of the pre-prodrug PR-104 into the treatment schedule provided significant additional activity, indicating proof-of-principle. Successful preclinical evaluation of a transferable gene that enables metabolism of both PET imaging agents (for vector visualisation) and prodrugs (for conditional enhancement of efficacy) is an important step towards the prospect of CDEPT entering clinical evaluation.

Efficient Secretion of Murine IL-2 From an Attenuated Strain of Clostridium sporogenes, a Novel Delivery Vehicle for Cancer Immunotherapy.

Despite a history dating back to the 1800s, using Clostridium bacteria to treat cancer has not advanced beyond the observation that they can colonise and partially destroy solid tumours. Progress has been hampered by their inability to eradicate the viable portion of tumours, and an instinctive anxiety around injecting patients with a bacterium whose close relatives cause tetanus and botulism. However, recent advances in techniques to genetically engineer Clostridium species gives cause to revisit this concept. This paper illustrates these developments through the attenuation of C. sporogenes to enhance its clinical safety, and through the expression and secretion of an immunotherapeutic. An 8.6 kb sequence, corresponding to a haemolysin operon, was deleted from the genome and replaced with a short non-coding sequence. The resultant phenotype of this strain, named C. sporogenes-NT, showed a reduction of haemolysis to levels similar to the probiotic strain, C. butyricum M588. Comparison to the parental strain showed no change in growth or sporulation. Following injection of tumour-bearing mice with purified spores of the attenuated strain, high levels of germination were detected in all tumours. Very low levels of spores and vegetative cells were detected in the spleen and lymph nodes. The new strain was transformed with four different murine IL-2-expressing plasmids, differentiated by promoter and signal peptide sequences. Biologically active mIL-2, recovered from the extracellular fraction of bacterial cultures, was shown to stimulate proliferation of T cells. With this investigation we propose a new, safer candidate for intratumoral delivery of cancer immunotherapeutics.

Prediction of individual speech recognition performance in complex listening conditions.

This study examined how well individual speech recognition thresholds in complex listening scenarios could be predicted by a current binaural speech intelligibility model. Model predictions were compared with experimental data measured for seven normal-hearing and 23 hearing-impaired listeners who differed widely in their degree of hearing loss, age, as well as performance in clinical speech tests. The experimental conditions included two masker types (multi-talker or two-talker maskers), and two spatial conditions (maskers co-located with the frontal target or symmetrically separated from the target). The results showed that interindividual variability could not be well predicted by a model including only individual audiograms. Predictions improved when an additional individual "proficiency factor" was derived from one of the experimental conditions or a standard speech test. Overall, the current model can predict individual performance relatively well (except in conditions high in informational masking), but the inclusion of age-related factors may lead to even further improvements.

Complete Genome Sequence of the Nonpathogenic Soil-Dwelling Bacterium Clostridium sporogenes Strain NCIMB 10696.

Clostridium sporogenes is a harmless spore-forming anaerobe that is widely distributed in soil/water and in the intestines of humans and animals. It is extensively used as a safe model to test the suitability of new preservative methods by the food industry and has potential to deliver therapeutic agents to tumors.

The potential of clostridial spores as therapeutic delivery vehicles in tumour therapy.

Despite substantial investment in prevention, treatment and aftercare, cancer remains a leading cause of death worldwide. More effective and accessible therapies are required. A potential solution is the use of endospore forming Clostridium species, either on their own, or as a tumour delivery vehicle for anti-cancer drugs. This is because intravenously injected spores of these obligate anaerobes can exclusively germinate in the hypoxic/necrotic regions present in solid tumours and nowhere else in the body. Research aimed at exploiting this unique phenomenon in anti-tumour strategies has been ongoing since the early part of the 20th century. Only in the last decade, however, has there been significant progress in the development and refinement of strategies based on spore-mediated tumour colonisation using a range of clostridial species. Much of this progress has been due to advances in genomics and our ability to modify strains using more sophisticated gene tools.

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.