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Cell Oct 2015The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors...
The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
Topics: Amino Acid Sequence; CRISPR-Cas Systems; Endonucleases; Francisella; Genetic Engineering; HEK293 Cells; Humans; Molecular Sequence Data; Nucleic Acid Conformation; RNA, Guide, CRISPR-Cas Systems; Sequence Alignment
PubMed: 26422227
DOI: 10.1016/j.cell.2015.09.038 -
Frontiers in Immunology 2023Immune checkpoint inhibitors have had a major impact on cancer treatment. Gut microbiota plays a major role in the cancer microenvironment, affecting treatment response....
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 and were relatively common in the group showing efficacy towards the anti-PD-1 antibody treatment (effective group). The proportions of (P = 0.022) and (P = 0.049) were significantly higher in the effective group than in the ineffective group. In addition, the proportion of (P = 0.033) was significantly higher in the ineffective group. Next, they were divided into irAE and non-irAE groups. The proportions of (P = 0.001) and (P = 0.001) were significantly higher in the group with irAEs than in those without, while the proportions of (P = 0.013) and the unclassified (P = 0.027) were significantly higher in the group without irAEs than those with. Furthermore, within the Effective group, and (both P = 0.001) were more abundant in the subgroup with irAEs than in those without them. In contrast, (P = 0.021) and (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.
Topics: Humans; Immune Checkpoint Inhibitors; Acidaminococcus; Neoplasms; Immunotherapy; Tumor Microenvironment
PubMed: 37207204
DOI: 10.3389/fimmu.2023.1164724 -
Nature Biotechnology Mar 2019Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM). To address this...
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM). To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
Topics: Acidaminococcus; Bacterial Proteins; CRISPR-Cas Systems; Endonucleases; Epigenesis, Genetic; Gene Editing; HEK293 Cells; Humans; Mutation; Ribonucleoproteins; T-Lymphocytes
PubMed: 30742127
DOI: 10.1038/s41587-018-0011-0 -
International Journal of Systematic and... Feb 2023The human gastrointestinal tract is inhabited by various microorganisms, including thousands of bacterial taxa that have yet to be cultured and characterized. In this...
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 sp. nov. (type strain NSJ-142=CGMCC 1.17903=KCTC 25346), sp. nov. (type strain NSJ-176=CGMCC 1.17933=KCTC 25355), gen. nov. sp. nov. (type strain NSJ-141=CGMCC 1.17902=KCTC 25345), gen. nov. sp. nov. (type strain NSJ-153=CGMCC 1.17915=KCTC 25350) and sp. nov. (type strain NSJ-152=CGMCC 1.17914=KCTC 25349).
Topics: Humans; Fatty Acids; Acidaminococcus; Phylogeny; DNA, Bacterial; RNA, Ribosomal, 16S; Base Composition; Bacterial Typing Techniques; Sequence Analysis, DNA; Firmicutes; Actinobacteria; Tenericutes; Feces; Phospholipids
PubMed: 36735588
DOI: 10.1099/ijsem.0.005648 -
Nature Biotechnology Jan 2021Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript,...
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.
Topics: Acidaminococcus; Apoptosis; Bacterial Proteins; CRISPR-Associated Protein 9; CRISPR-Associated Proteins; CRISPR-Cas Systems; Cell Line, Tumor; Endodeoxyribonucleases; Gene Editing; Gene Library; HEK293 Cells; Humans; RNA, Guide, CRISPR-Cas Systems
PubMed: 32661438
DOI: 10.1038/s41587-020-0600-6 -
FEMS Microbiology Letters Apr 2019The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a...
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.
Topics: Acidaminococcus; Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; Catalytic Domain; DNA Cleavage; Endodeoxyribonucleases; Endonucleases; Escherichia coli; Gene Editing; HEK293 Cells; Humans; Nucleotide Motifs; Plasmids
PubMed: 31004485
DOI: 10.1093/femsle/fnz085 -
Nutrients Dec 2021Whether the gut microbiome in obesity is characterized by lower diversity and altered composition at the phylum or genus level may be more accurately investigated using... (Meta-Analysis)
Meta-Analysis
Whether the gut microbiome in obesity is characterized by lower diversity and altered composition at the phylum or genus level may be more accurately investigated using high-throughput sequencing technologies. We conducted a systematic review in PubMed and Embase including 32 cross-sectional studies assessing the gut microbiome composition by high-throughput sequencing in obese and non-obese adults. A significantly lower alpha diversity (Shannon index) in obese versus non-obese adults was observed in nine out of 22 studies, and meta-analysis of seven studies revealed a non-significant mean difference (-0.06, 95% CI -0.24, 0.12, = 81%). At the phylum level, significantly more Firmicutes and fewer Bacteroidetes in obese versus non-obese adults were observed in six out of seventeen, and in four out of eighteen studies, respectively. Meta-analyses of six studies revealed significantly higher Firmicutes (5.50, 95% 0.27, 10.73, = 81%) and non-significantly lower Bacteroidetes (-4.79, 95% CI -10.77, 1.20, = 86%). At the genus level, lower relative proportions of and and higher , , , , , , , , , , , , and were found in obese versus non-obese adults. Although a proportion of studies found lower diversity and differences in gut microbiome composition in obese versus non-obese adults, the observed heterogeneity across studies precludes clear answers.
Topics: Bacteria; Feces; Gastrointestinal Microbiome; High-Throughput Nucleotide Sequencing; Humans; Obesity
PubMed: 35010887
DOI: 10.3390/nu14010012 -
New Microbes and New Infections Sep 2019strain Marseille-P4266 (= CSURP4266) is a new species isolated from a fresh human stool specimen.
strain Marseille-P4266 (= CSURP4266) is a new species isolated from a fresh human stool specimen.
PubMed: 31341626
DOI: 10.1016/j.nmni.2019.100573 -
Nature Methods Sep 2019The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular...
The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.
Topics: Acidaminococcus; CRISPR-Cas Systems; Endonucleases; Gene Editing; Gene Regulatory Networks; Genetic Engineering; Genome, Human; HEK293 Cells; Humans; Plasmids; RNA, Guide, CRISPR-Cas Systems; Transcriptional Activation
PubMed: 31406383
DOI: 10.1038/s41592-019-0508-6 -
Cell May 2016Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of...
Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox.
Topics: Acidaminococcus; Bacterial Proteins; Crystallography, X-Ray; DNA; Genetic Techniques; Models, Molecular; Nucleic Acid Heteroduplexes; RNA, Guide, CRISPR-Cas Systems
PubMed: 27114038
DOI: 10.1016/j.cell.2016.04.003