Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.

Recapitulating familial hypercholesterolemia in a mouse model by knock-in patient-specific LDLR mutation.

Familial hypercholesterolemia (FH) is one of the most prevalent monogenetic disorders leading to cardiovascular disease (CVD) worldwide. Mutations in Ldlr, encoding a membrane-spanning protein, account for the majority of FH cases. No effective and safe clinical treatments are available for FH. Adenine base editor (ABE)-mediated molecular therapy is a promising therapeutic strategy to treat genetic diseases caused by point mutations, with evidence of successful treatment in mouse disease models. However, due to the differences in the genomes between mice and humans, ABE with specific sgRNA, a key gene correction component, cannot be directly used to treat FH patients. Thus, we generated a knock-in mouse model harboring the partial patient-specific fragment and including the Ldlr W490X mutation. LdlrW490X/W490X mice recapitulated cholesterol metabolic disorder and clinical manifestations of atherosclerosis associated with FH patients, including high plasma low-density lipoprotein cholesterol levels and lipid deposition in aortic vessels. Additionally, we showed that the mutant Ldlr gene could be repaired using ABE with the cellular model. Taken together, these results pave the way for ABE-mediated molecular therapy for FH.

Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.

New insights into the influence of pre-culture on robust solvent production of C. acetobutylicum.

Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured. KEY POINTS: • Both cryo- and pre-culture strongly impact the cultivation of C. acetobutylicum • Cultivation conditions of the pre-culture are a reason for the acid crash • Inoculum from cells in stationary growth phase ensures shift to solventogenesis.

The effect of target detection task on memory encoding varies in different stimulus onset asynchronies.

The attentional boost effect (ABE) and action-induced memory enhancement (AIME) suggest that memory performance for target-paired items is superior to that for distractor-paired items when participants performed a target detection task and a memory encoding task simultaneously. Though the memory enhancement has been well established, the temporal dynamics of how the target detection task influenced memory encoding remains unclear. To investigate this, we manipulated the stimulus onset asynchrony (SOA) between detection stimuli and the words to be memorized using a remember/know study-test paradigm, and we focused primarily on memory performance for the words that appeared after the detection response. The results showed that target-paired memory enhancement was robust from SOA = 0 s to SOA = 0.75 s, but was not significant when examined by itself in Experiment 1A or weakened in Experiment 2 and the conjoint analysis when SOA = 1 s, which were only observed in R responses. The post-response memory enhancement still existed when there was no temporal overlap between the word and target, similar to the magnitude of memory enhancement observed with temporal overlap. These results supported the view that target-paired memory enhancement (recollection rather than familiarity) occurred irrespective of whether the items appeared simultaneously with the targets or within a short period after the response, and the temporal overlap of the word and target was not necessary for post-response memory enhancement.

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope.

BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797-C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.

BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.

The Effect of Technological Conditions on ABE Fermentation and Butanol Production of Rye Straw and the Composition of Volatile Compounds.

The objective of this study was to evaluate the effect of pretreatment and different technological conditions on the course of ABE fermentation of rye straw (RS) and the composition of volatile compounds in the distillates obtained. The highest concentration of ABE and butanol was obtained from the fermentation of pretreated rye straw by alkaline hydrolysis followed by detoxification and enzymatic hydrolysis. After 72 h of fermentation, the maximum butanol concentration, productivity, and yield from RS were 16.11 g/L, 0.224 g/L/h, and 0.402 g/g, respectively. Three different methods to produce butanol were tested: the two-step process (SHF), the simultaneous process (SSF), and simultaneous saccharification with ABE fermentation (consolidation SHF/SSF). The SHF/SSF process observed that ABE concentration (21.28 g/L) was higher than in the SSF (20.03 g/L) and lower compared with the SHF (22.21 g/L). The effect of the detoxification process and various ABE fermentation technologies on the composition of volatile compounds formed during fermentation and distillation were analyzed.

Mathematical modeling of ABE fermentation on glucose substrate with Zn supplementation for enhanced butanol production.

Supplementation or limitation of some micronutrients during acetone-butanol-ethanol (ABE) fermentation has led to improvement in butanol yield and productivity. A mechanistic model of ABE fermentation offers insights in understanding these complex interactions and improving productivity through optimal culture conditions. This study proposes a mechanistic kinetic model of ABE fermentation by two Clostridium Acetobutylicum strains, L7 and ATCC 824 using glucose as sole carbon source without zinc and with various zinc doses. The model incorporates enzyme regulation by zinc on several glycolytic, acidogenesis and solventogenesis enzymes. The model was fitted and validated to experimental data collected from the published literature. The simulated results were in compliance with the experimental data, most importantly indicating higher glucose consumption and butanol productivity when supplemented with zinc compared to the control culture. The average squared correlation factor (R2) between the experimental and the simulated results, without and with zinc, were 0.99 and 0.96 for glucose, and 0.89 and 0.95 for butanol, respectively. A sensitivity analysis performed on the fitted and validated model indicated that the glucose consumption and growth parameters most influenced the model outputs. The developed model can be used as a template for modeling ABE fermentation under different combinations of micronutrients that may offer improved butanol yield and productivity.

A precise and efficient adenine base editor.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

The differential value of radiomics based on traditional T1-weighted sequences in newborns with hyperbilirubinemia.

BACKGROUND: On the basis of visual-dependent reading method, radiological recognition and assessment of neonatal hyperbilirubinemia (NH) or acute bilirubin encephalopathy (ABE) on conventional magnetic resonance imaging (MRI) sequences are challenging. Prior studies had shown that radiomics was possible to characterize ABE-induced intensity and morphological changes on MRI sequences, and it has emerged as a desirable and promising future in quantitative and objective MRI data extraction. To investigate the utility of radiomics based on T1-weighted sequences for identifying neonatal ABE in patients with hyperbilirubinemia and differentiating between those with NH and the normal controls. METHODS: A total of 88 patients with NH were enrolled, including 50 patients with ABE and 38 ABE-negative individuals, and 70 age-matched normal neonates were included as controls. All participants were divided into training and validation cohorts in a 7:3 ratio. Radiomics features extracted from the basal ganglia of T1-weighted sequences on magnetic resonance imaging were evaluated and selected to set up the prediction model using the K-nearest neighbour-based bagging algorithm. A receiver operating characteristic curve was plotted to assess the differentiating performance of the radiomics-based model. RESULTS: Four of 744 radiomics features were selected for the diagnostic model of ABE. The radiomics model yielded an area under the curve (AUC) of 0.81 and 0.82 in the training and test cohorts, with accuracy, precision, sensitivity, and specificity of 0.82, 0.80, 0.91, and 0.69 and 0.78, 0.8, 0.8, and 0.75, respectively. Six radiomics features were selected in this model to distinguish those with NH from the normal controls. The AUC for the training cohort was 0.97, with an accuracy of 0.92, a precision of 0.92, a sensitivity of 0.93, and a specificity of 0.90. The performance of the radiomics model was confirmed by testing the test cohort, and the AUC, accuracy, precision, sensitivity, and specificity were 0.97, 0.92, 0.96, 0.89, and 0.95, respectively. CONCLUSIONS: The proposed radiomics model based on traditional TI-weighted sequences may be used effectively for identifying ABE and even differentiating patients with NH from the normal controls, which can provide microcosmic information beyond experience-dependent vision and potentially assist in clinical diagnosis and treatment.

Blockchain-Based Data Access Control and Key Agreement System in IoT Environment.

Recently, with the increasing application of the Internet of Things (IoT), various IoT environments such as smart factories, smart homes, and smart grids are being generated. In the IoT environment, a lot of data are generated in real time, and the generated IoT data can be used as source data for various services such as artificial intelligence, remote medical care, and finance, and can also be used for purposes such as electricity bill generation. Therefore, data access control is required to grant access rights to various data users in the IoT environment who need such IoT data. In addition, IoT data contain sensitive information such as personal information, so privacy protection is also essential. Ciphertext-policy attribute-based encryption (CP-ABE) technology has been utilized to address these requirements. Furthermore, system structures applying blockchains with CP-ABE are being studied to prevent bottlenecks and single failures of cloud servers, as well as to support data auditing. However, these systems do not stipulate authentication and key agreement to ensure the security of the data transmission process and data outsourcing. Accordingly, we propose a data access control and key agreement scheme using CP-ABE to ensure data security in a blockchain-based system. In addition, we propose a system that can provide data nonrepudiation, data accountability, and data verification functions by utilizing blockchains. Both formal and informal security verifications are performed to demonstrate the security of the proposed system. We also compare the security, functional aspects, and computational and communication costs of previous systems. Furthermore, we perform cryptographic calculations to analyze the system in practical terms. As a result, our proposed protocol is safer against attacks such as guessing attacks and tracing attacks than other protocols, and can provide mutual authentication and key agreement functions. In addition, the proposed protocol is more efficient than other protocols, so it can be applied to practical IoT environments.

An Access Control Scheme Based on Blockchain and Ciphertext Policy-Attribute Based Encryption.

Ciphertext policy-attribute-based encryption (CP-ABE), which provides fine-grained access control and ensures data confidentiality, is widely used in data sharing. However, traditional CP-ABE schemes often choose to outsource data to untrusted third-party cloud service providers for storage or to verify users' access rights through third parties, which increases the risk of privacy leakage and also suffers from the problem of opaque permission verification. This paper proposes an access control scheme based on blockchain and CP-ABE, which is based on multiple authorization centers and supports policy updating. In addition, blockchain technology's distributed, decentralized, and tamper-proof features are utilized to solve the trust crisis problem in the data-sharing process. Security analysis and performance evaluation show that the proposed scheme improves the computational efficiency by 18%, 26%, and 68% compared to previous references. The proposed scheme also satisfies the indistinguishability under chosen-plaintext attack (IND-CPA).

dCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.

A Lightweight CP-ABE Scheme with Direct Attribute Revocation for Vehicular Ad Hoc Network.

Ciphertext-Policy Attribute-Based Encryption (CP-ABE) technology provides a new solution to address the security and fine-grained access control of traffic information in vehicular ad hoc networks (VANETs). However, in most CP-ABE schemes for VANETs, attribute revocation suffers from high system consumption and complex revocation operations, as well as from high computational overhead and low efficiency due to the use of bilinear pairwise operations. Based on this, this paper proposes a lightweight CP-ABE scheme that supports direct attribute revocation in VANETs. The scheme implements an agent-based direct attribute revocation mechanism by separating dynamic and static attributes of vehicle terminals, which reduces system consumption and simplifies the revocation operation process. The scheme uses scalar multiplication on elliptic curves instead of bilinear pairing operations and uses computational outsourcing techniques to reduce the terminal decryption cost and improve the efficiency of the scheme. The security and performance analysis shows that the overall efficiency of our scheme is better than the existing schemes under the premise of ensuring data confidentiality and integrity.

A privacy-preserving blockchain-based tracing model for virus-infected people in cloud.

The outbreak of COVID-19 has exposed the privacy of positive patients to the public, which will lead to violations of users' rights and even threaten their lives. A privacy-preserving scheme involving virus-infected positive patients is proposed by us. The traditional ciphertext policy attribute-based encryption (CP-ABE) has the features of enhanced plaintext security and fine-grained access control. However, the encryption process requires the high computational performance of the device, which puts a high strain on resource-limited devices. After semi-honest users successfully decrypt the data, they will get the real private data, which will cause serious privacy leakage problems. Traditional cloud-based data management architectures are extremely vulnerable in the face of various cyberattacks. To address the above challenges, a verifiable ABE scheme based on blockchain and local differential privacy is proposed, using LDP to perturb the original data locally to a certain extent to resist collusion attacks, outsourcing encryption and decryption to corresponding service providers to reduce the pressure on mobile terminals, and deploying smart contracts in combination with blockchain for fair execution by all parties to solve the problem of returning wrong search results in a semi-honest cloud server. Detailed security proofs are performed through the defined security goals, which shows that the proposed scheme is indeed privacy-protective. The experimental results show that the scheme is optimized in terms of data accuracy, computational overhead, storage performance, and fairness. In terms of efficiency, it greatly reduces the local load, enhances personal privacy protection, and has high practicality as well as reliability. As far as we know, it is the first case of applying the combination of LDP technology and blockchain to a tracing system, which not only mitigates poisoning attacks on user data, but also improves the accuracy of the data, thus making it easier to identify infected contacts and making a useful contribution to health prevention and control efforts.

Online/Offline MA-CP-ABE with Cryptographic Reverse Firewalls for IoT.

Devices in the Internet of Things (IoT) usually use cloud storage and cloud computing to save storage and computing cost. Therefore, the efficient realization of one-to-many communication of data on the premise of ensuring the security of cloud storage data is a challenge. Ciphertext-Policy Attribute-Based Encryption (CP-ABE) can not only protect the security of data in the cloud and achieve one-to-many communication but also achieve fine-grained access control for data. However, the single-authority CP-ABE faces the crisis of single point of failure. In order to improve security, the Multi-Authority CP-ABE (MA-CP-ABE) is adopted. Although there are provably-secure MA-CP-ABE schemes, Edward Snowden's research shows that provably-secure cryptographic schemes are vulnerable to backdoor attacks, resulting in secret disclosure, and thus threatening security. In addition, ABE requires huge computational overhead in key generation, encryption and decryption, which increase with the increase in the number of attributes and the complexity of the access structure, and there are a large number of resource-constrained devices in the IoT. To mitigate this issue, we construct the Online/Offline MA-CP-ABE with Cryptographic Reverse Firewalls (OO-MA-CP-ABE-CRFs) scheme. This scheme not only uses Cryptographic Reverse Firewall (CRF) to resist backdoor attacks but also uses online/offline key generation, online/offline encryption and outsourcing encryption technology to optimize the efficiency of the MA-CP-ABE scheme with reverse firewall, reducing the storage and computing cost of users. Finally, the security of the OO-MA-CP-ABE-CRFs scheme is proved, and the experimental results indicate that the scheme is efficient and practical.

Delivery of Cas9-guided ABE8e into stem cells using poly(l-lysine) polypeptides for correction of the hemophilia-associated FIX missense mutation.

The coagulation factor 9 gene (FIX) point mutation contributes to most hemophilia B cases, providing ideal gene correction models. Here we identified the frequent mutation G20519A (R226Q) in FIX, which resulted in many severe and moderate hemophilia B patients. This study aimed to investigate the effect of HDR and base editing in correcting FIX mutant. We first constructed HEK293 and liver-derived cell lines Huh7 cells stabling carrying mutated FIX containing G20519A (HEK293-FIXmut and Huh7-FIXmut). Then, CRISPR/Cas9-based homology-directed repair (HDR) and base editing were used for the correction of this mutated point. We used Cas9 nickase (nCas9) mediated HDR and the advanced base editor ABE8e to correct G20519A and then measured the concentration and activity of FIX. Furthermore, we used the star-shaped poly(lysine) gene nanocarriers to deliver the ABE8e correction systems into HEK293-FIXmut and Huh7-FIXmut stem cells to correct mutated FIX. As a result, we found that gRNAs directed inefficient HDR in correcting G20519A. The ABE8e corrected the mutation efficiently in both HEK293-FIXmut and Huh7-FIXmut stem cells. In addition, the star-shaped poly(lysine) carriers delivered non-viral vectors into stem cells efficiently. The nanocarriers-delivered ABE8e system corrected mutated FIX in stem cells, and the stem cells secreted active FIX in high concentration. In conclusion, our study provides a potential alternative for correcting mutated FIX in hemophilia B patients.

Reclassification of Clostridium aurantibutyricum Hellinger 1944 and Clostridium roseum (ex McCoy and McClung 1935) Cato et al. 1988.

Clostridium aurantibutyricum, Clostridium felsineum and Clostridium roseum share a very high similarity based on multi-locus sequence analysis. In this study, their correct taxonomic status was determined using genomic and phenotypic investigations. Average nucleotide identity based on MUMmer alignment of the genomes and in silico DNA-DNA hybridization resulted in values of 98.55-100 and 78.7-100 %, respectively, strongly indicating that all strains are members of the same species. In addition, morphological investigations, fatty acid analyses and substrate utilization tests revealed no striking differences between the strains. Therefore, we propose the reclassification of C. aurantibutyricum and C. roseum as later heterotypic synonyms of C. felsineum. The type strain is lodged in several culture collections (ATCC 17788T=DSM 794T=NCIMB 10690T).

Identification of serine/threonine kinases that regulate metabolism and sporulation in Clostridium beijerinckii.

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2 g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation.