Turicibacter and Acidaminococcus predict immune-related adverse events and efficacy of immune checkpoint inhibitor.

Introduction: Immune checkpoint inhibitors have had a major impact on cancer treatment. Gut microbiota plays a major role in the cancer microenvironment, affecting treatment response. The gut microbiota is highly individual, and varies with factors, such as age and race. Gut microbiota composition in Japanese cancer patients and the efficacy of immunotherapy remain unknown. Methods: We investigated the gut microbiota of 26 patients with solid tumors prior to immune checkpoint inhibitor monotherapy to identify bacteria involved in the efficacy of these drugs and immune-related adverse events (irAEs). Results: The genera Prevotella and Parabacteroides were relatively common in the group showing efficacy towards the anti-PD-1 antibody treatment (effective group). The proportions of Catenibacterium (P = 0.022) and Turicibacter (P = 0.049) were significantly higher in the effective group than in the ineffective group. In addition, the proportion of Desulfovibrion (P = 0.033) was significantly higher in the ineffective group. Next, they were divided into irAE and non-irAE groups. The proportions of Turicibacter (P = 0.001) and Acidaminococcus (P = 0.001) were significantly higher in the group with irAEs than in those without, while the proportions of Blautia (P = 0.013) and the unclassified Clostridiales (P = 0.027) were significantly higher in the group without irAEs than those with. Furthermore, within the Effective group, Acidaminococcus and Turicibacter (both P = 0.001) were more abundant in the subgroup with irAEs than in those without them. In contrast, Blautia (P = 0.021) and Bilophila (P= 0.033) were statistically significantly more common in those without irAEs. Discussion: Our Study suggests that the analysis of the gut microbiota may provide future predictive markers for the efficacy of cancer immunotherapy or the selection of candidates for fecal transplantation for cancer immunotherapy.

Acidaminococcus homini s sp. nov., Amedibacillus hominis sp. nov., Lientehia hominis gen. nov. sp. nov., Merdimmobilis hominis gen. nov. sp. nov., and Paraeggerthella hominis sp. nov., isolated from human faeces.

The human gastrointestinal tract is inhabited by various microorganisms, including thousands of bacterial taxa that have yet to be cultured and characterized. In this report, we describe the isolation, cultivation, genotypic and phenotypic characterization and taxonomy of five novel anaerobic bacterial strains that were recovered during the massive cultivation and isolation of gut microbes from human faecal samples. On the basis of the polyphasic taxonomic results, we propose two novel genera and five novel species. They are Acidaminococcus hominis sp. nov. (type strain NSJ-142T=CGMCC 1.17903T=KCTC 25346T), Amedibacillus hominis sp. nov. (type strain NSJ-176T=CGMCC 1.17933T=KCTC 25355T), Lientehia hominis gen. nov. sp. nov. (type strain NSJ-141T=CGMCC 1.17902T=KCTC 25345T), Merdimmobilis hominis gen. nov. sp. nov. (type strain NSJ-153T=CGMCC 1.17915T=KCTC 25350T) and Paraeggerthella hominis sp. nov. (type strain NSJ-152T=CGMCC 1.17914T=KCTC 25349T).

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.

BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Optimization of AsCas12a for combinatorial genetic screens in human cells.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.

IL-13 mRNA Tissue Content Identifies Two Subsets of Adult Ulcerative Colitis Patients With Different Clinical and Mucosa-Associated Microbiota Profiles.

BACKGROUND AND AIMS: A personalized approach to therapy hold great promise to improve disease outcomes. To this end, the identification of different subsets of patients according to the prevalent pathogenic process might guide the choice of therapeutic strategy. We hypothesize that ulcerative colitis [UC] patients might be stratified according to distinctive cytokine profiles and/or to a specific mucosa-associated microbiota. METHODS: In a cohort of clinically and endoscopic active UC patients and controls, we used quantitative PCR to analyse the mucosal cytokine mRNA content and 16S rRNA gene sequencing to assess the mucosa-associated microbiota composition. RESULTS: We demonstrate, by means of data-driven approach, the existence of a specific UC patient subgroup characterized by elevated IL-13 mRNA tissue content separate from patients with low IL-13 mRNA tissue content. The two subsets differ in clinical-pathological characteristics. High IL-13 mRNA patients are younger at diagnosis and have a higher prevalence of extensive colitis than low IL-13 mRNA patients. They also show more frequent use of steroid/immunosuppressant/anti-tumour necrosis factor α therapy during 1 year of follow-up. The two subgroups show differential enrichment of mucosa-associated microbiota genera with a prevalence of Prevotella in patients with high IL-13 mRNA tissue content and Sutterella and Acidaminococcus in patients with low IL-13 mRNA tissue content. CONCLUSION: Assessment of mucosal IL-13 mRNA might help in the identification of a patient subgroup that might benefit from a therapeutic approach modulating IL-13. PODCAST: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

Rosacea and its association with enteral microbiota in Korean females.

Rosacea is a chronic inflammatory dermatosis affecting the face and eyes. An association between systemic comorbidities and rosacea has been reported, but the link to enteral microbiota is uncertain. We aimed to investigate the link between rosacea and enteral microbiota. A cross-sectional study was performed in a sample of Korean women who participated in a health check-up programme at the Kangbuk Samsung Hospital Health Screening Center between 23 June 2014 and 5 September 2014. The gut microbiome was evaluated by 16S rRNA gene and metagenome sequence analyses. A total of 12 rosacea patients and 251 controls were enrolled. We identified links between rosacea and several changes in gut microbiota: reduced abundance of Peptococcaceae family unknown genus, Methanobrevibacter (genus), Slackia (genus), Coprobacillus (genus), Citrobacter (genus), and Desulfovibrio (genus), and increased abundance of Acidaminococcus (genus), Megasphaera (genus), and Lactobacillales order unknown family unknown genus. A link between rosacea and enteral microbiota was observed in this metagenomic study. A large and elaborate study is needed to confirm these findings and to elucidate the mechanisms involved.

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.

Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells.

BACKGROUND: Cpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified. RESULTS: Here, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells. Exploiting a plasmid that contains two GFP halves with overlapping sequences and exploring 20 targets, on all but one both nucleases consistently performed with above 10 % efficiency. Several Cas9 nucleases have been previously characterised in order to find an orthogonal counterpart for the most widely used promiscuous SpCas9. Here, we found that AsCpf1 and LbCpf1 might be better candidates than three of the best such counterparts: Cas9 from Staphylococcus aureus, from Streptococcus thermophilus and from Neisseria meningitidis, when assessed for inducing efficient SSA mediated repair in N2a cells. When tested on genomic targets exploiting HR, both nucleases were able to induce the integration of a donor cassette with 1000 bp-long homologous arms. We also generated plasmids that express these Cpf1 nucleases together with their cognate crRNAs and that are equipped with type IIS restriction enzyme sites to facilitate spacer cloning. CONCLUSIONS: Our results suggest that employing As- or LbCpf1 nuclease to induce homology directed repair in N2a cells, although is less effective at present than employing SpCas9, it is an equally or more effective tool than the most frequently used orthogonal Cas9 counterparts of SpCas9. These findings support the position of Cpf1 nucleases on the side of SpCas9 on the palette of effective genome engineering tools. REVIEWERS: This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.