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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 -
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 -
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 -
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 -
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 -
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 -
Angewandte Chemie (International Ed. in... Nov 2019An 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...
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 β1 (TGF-β1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.
Topics: Acidaminococcus; Aptamers, Nucleotide; Biosensing Techniques; CRISPR-Cas Systems; DNA Cleavage; DNA, Viral; Electrochemical Techniques; Electrodes; Human papillomavirus 16; Humans; Immobilized Nucleic Acids; Limit of Detection; Mesenchymal Stem Cells; Parvovirus; Sensitivity and Specificity; Surface Properties; Transforming Growth Factor beta1
PubMed: 31568601
DOI: 10.1002/anie.201910772 -
Nature Communications May 2022Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs....
Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.
Topics: Acidaminococcus; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Staphylococcus aureus; Streptococcus pyogenes
PubMed: 35513429
DOI: 10.1038/s41467-022-30196-9 -
Frontiers in Medicine 2022Most colorectal cancer (CRC) cases are sporadic and develop along the adenoma-carcinoma sequence. Intestinal microbial dysbiosis is involved in the development of...
BACKGROUND
Most colorectal cancer (CRC) cases are sporadic and develop along the adenoma-carcinoma sequence. Intestinal microbial dysbiosis is involved in the development of colorectal cancer. However, there are still no absolute markers predicting the progression from adenoma to carcinoma. This study aimed to investigate the characteristics of intestinal microbiota in patients with colorectal adenoma and carcinoma and its correlations with clinical characteristics.
METHODS
Fecal samples were collected from 154 patients with CRC, 20 patients with colorectal adenoma (AD) and 199 healthy controls. To analyze the differences in the intestinal microbiota, 16S rRNA gene sequencing was conducted.
RESULTS
At the genus level, there were four significantly different genera among the three groups, namely Acidaminococcus, Alloprevotella, Mycoplasma, and Sphingobacterium, while Acidaminococcus significantly decreased with the order of Control-AD-CRC ( < 0.05). In addition, Parvimonas, Peptostreptococcus, Prevotella, Butyricimonas, Alistipes, and Odoribacter were the key genera in the network of colorectal adenoma/carcinoma-associated bacteria. The top 10 most important species, including , , , , , , , , and , showed the best performance in distinguishing AD from CRC (AUC = 85.54%, 95% CI: 78.83-92.25%). The clinicopathologic features, including age, gender, tumor location, differentiation degree, and TNM stage, were identified to be closely linked to the intestinal microbiome in CRC.
CONCLUSION
Several intestinal bacteria changed along the adenoma-carcinoma sequence and might be the potential markers for the diagnosis and treatment of colorectal adenoma/carcinoma. Intestinal microbiota characteristics in CRC should account for the host factors.
PubMed: 35935780
DOI: 10.3389/fmed.2022.888340 -
Frontiers in Cellular and Infection... 2022This is the first study on gut microbiota (GM) in children affected by coronavirus disease 2019 (COVID-19). Stool samples from 88 patients with suspected severe acute...
This is the first study on gut microbiota (GM) in children affected by coronavirus disease 2019 (COVID-19). Stool samples from 88 patients with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 95 healthy subjects were collected (admission: 3-7 days, discharge) to study GM profile by 16S rRNA gene sequencing and relationship to disease severity. The study group was divided in COVID-19 (68), Non-COVID-19 (16), and MIS-C (multisystem inflammatory syndrome in children) (4). Correlations among GM ecology, predicted functions, multiple machine learning (ML) models, and inflammatory response were provided for COVID-19 and Non-COVID-19 cohorts. The GM of COVID-19 cohort resulted as dysbiotic, with the lowest α-diversity compared with Non-COVID-19 and CTRLs and by a specific β-diversity. Its profile appeared enriched in , , and and reduced in , , , , , , and , compared with CTRLs ( 0.05). All GM paired-comparisons disclosed comparable results through all time points. The comparison between COVID-19 and Non-COVID-19 cohorts highlighted a reduction of in the COVID-19 cohort ( < 0.05). The GM of MIS-C cohort was characterized by an increase of , , , , and and a decrease of , , , and , compared with CTRLs. Stratifying for disease severity, the GM associated to "moderate" COVID-19 was characterized by lower α-diversity compared with "mild" and "asymptomatic" and by a GM profile deprived in , , , and and enriched in , , , , , , and The ML models identified , , , , , , , , , , , , , , , , and as microbial markers of COVID-19. The KEGG ortholog (KO)-based prediction of GM functional profile highlighted 28 and 39 KO-associated pathways to COVID-19 and CTRLs, respectively. Finally, and correlated with proinflammatory cytokines regardless disease severity. Unlike adult GM profiles, was a specific marker of pediatric COVID-19 GM. The durable modification of patients' GM profile suggested a prompt GM quenching response to SARS-CoV-2 infection since the first symptoms. and reduced fatty acid and amino acid degradation were proposed as specific COVID-19 disease traits, possibly associated to restrained severity of SARS-CoV-2-infected children. Altogether, this evidence provides a characterization of the pediatric COVID-19-related GM.
Topics: Adult; Bacteroides; Bifidobacterium; COVID-19; Child; Clostridium; Feces; Gastrointestinal Microbiome; Humans; RNA, Ribosomal, 16S; SARS-CoV-2; Systemic Inflammatory Response Syndrome
PubMed: 35873161
DOI: 10.3389/fcimb.2022.908492