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International Journal of Molecular... Jan 2022The efficiency of the RNA-guided Cas12a nuclease of sp. was compared with Cas9 from , for functional genomics in . We deployed optimized conditions for the ratio of...
The efficiency of the RNA-guided Cas12a nuclease of sp. was compared with Cas9 from , for functional genomics in . We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. Cas12a was more efficient than Cas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With Cas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the Cas9 plus ssODN, Cas12a plus NT-ssODN, and Cas12a plus T-ssODN groups, respectively. -cleavage against the ssODNs by activated Cas12a was not apparent in vitro. Cas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although Cas12a induced fewer mutations per genome than Cas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.
Topics: Animals; Base Sequence; CRISPR-Cas Systems; Catalysis; Female; Gene Dosage; Gene Knockout Techniques; Genes, Protozoan; Genetic Loci; Humans; Mutation; Oligonucleotides; RNA, Guide, CRISPR-Cas Systems; Recombinational DNA Repair; Reference Standards; Ribonucleases; Schistosoma mansoni; Transcription, Genetic; Transgenes
PubMed: 35054816
DOI: 10.3390/ijms23020631 -
Frontiers in Oral Health 2021Inflammation is a driven force in modulating microbial communities, but little is known about the interplay between colonizing microorganisms and the immune response in...
Inflammation is a driven force in modulating microbial communities, but little is known about the interplay between colonizing microorganisms and the immune response in periodontitis. Since local and systemic inflammation may play a whole role in disease, we aimed to evaluate the oral and fecal microbiome of patients with periodontitis and to correlate the oral microbiome data with levels of inflammatory mediator in saliva. Nine patients with periodontitis (P) in Stage 3/Grade B and nine age-matched non-affected controls (H) were evaluated. Microbial communities of oral biofilms (the supra and subgingival from affected and non-affected sites) and feces were determined by sequencing analysis of the V3-V4 region. Salivary levels of 40 chemokines and cytokines were correlated with oral microbiome data. Supragingival microbial communities of P differed from H (Pielou's evenness index, and Beta diversity, and weighted UniFrac), since relative abundance (RA) of was higher in P, whereas and were more abundant in H. Subgingival non-affected sites of P did not differ from H, except for a lower abundance of . The microbiome of affected periodontitis sites (PD ≥ 4 mm) clustered apart from the subgingival sites of H. Oral pathobionts was more abundant in sub and supragingival biofilms of P than H. Fecal samples of P were enriched with when compared to H. The salivary levels of interleukin 6 (IL-6) and inflammatory chemokines were positively correlated with the RA of several recognized and putative pathobionts, whereas the RA of beneficial species, such as and was negatively correlated with the levels of Chemokine C-C motif Ligand 2 (CCL2), which is considered protective. Dysbiosis in patients with periodontitis was not restricted to periodontal pockets but was also seen in the supragingival and subgingival non-affected sites and feces. Subgingival dysbiosis revealed microbial signatures characteristic of different immune profiles, suggesting a role for candidate pathogens and beneficial organisms in the inflammatory process of periodontitis.
PubMed: 35048045
DOI: 10.3389/froh.2021.722495 -
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 -
Nucleic Acids Research Jan 2022CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties....
CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.
Topics: Acidaminococcus; Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA; DNA Cleavage; Endodeoxyribonucleases; Francisella; Gene Editing
PubMed: 34951459
DOI: 10.1093/nar/gkab1227 -
Nucleic Acids Research Jan 2022The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with...
The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5' of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.
Topics: Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; Endodeoxyribonucleases; Gene Editing; HEK293 Cells; Humans; Protein Splicing
PubMed: 34908140
DOI: 10.1093/nar/gkab1191 -
Journal of Animal Science and Technology Sep 2021Short-chain fatty acids (SCFAs) are metabolic products produced during the microbial fermentation of non-digestible fibers and play an important role in metabolic...
Short-chain fatty acids (SCFAs) are metabolic products produced during the microbial fermentation of non-digestible fibers and play an important role in metabolic homeostasis and overall gut health. In this study, we investigated the effects of supplementation with multispecies probiotics (MSPs) containing , , and on the gut microbiota, and fecal SCFAs and lactate levels of weaned pigs. A total of 38 pigs weaned at 4 weeks of age were fed either a basal diet or a diet supplemented with MSPs for 6 weeks. MSP administration significantly increased the fecal concentrations of lactate (2.3-fold; < 0.01), acetate (1.8-fold; < 0.05), and formate (1.4-fold; < 0.05). Moreover, MSP supplementation altered the gut microbiota of the pigs by significantly increasing the population of potentially beneficial bacteria such as , , , , and . MSP supplementation also decreased the abundance of pathogenic bacteria such as Escherichia and . The modulation of the gut microbiota was observed to be strongly correlated with the changes in fecal SCFAs and lactate levels. Furthermore, we found changes in the functional pathways present within the gut, which supports our findings that MSP modulates the gut microbiota and SCFAs levels in pigs. The results support the potential use of MSPs to improve the gut health of animals by modulating SCFAs production.
PubMed: 34796353
DOI: 10.5187/jast.2021.e94 -
Genetics Jan 2022The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes...
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is the most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from S. pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1) are orthogonal systems capable of operating simultaneously in zebrafish. CRISPR systems from Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) were also active in embryos. We implemented multichannel CRISPR recording using three CRISPR systems and show that LbaCas12a may provide superior information density compared with previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. Our results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.
Topics: Gene Editing
PubMed: 34735006
DOI: 10.1093/genetics/iyab196 -
Frontiers in Microbiology 2021Dietary fiber and flavonoids have substantial influence on the human gut microbiota composition that significantly impact health. Recent studies with dietary supplements...
Dietary fiber and flavonoids have substantial influence on the human gut microbiota composition that significantly impact health. Recent studies with dietary supplements such as quercetin and rice bran have shown beneficial impacts on the host alongside a positive influence of the gut microbiota. The specific bacterial species impacted by quercetin or rice bran in the diet is not well understood. In this study, we used a minibioreactor array system as a model to determine the effect of quercetin and rice bran individually, as well as in combination, on gut microbiota without the confounding host factors. We found that rice bran exerts higher shift in gut microbiome composition when compared to quercetin. At the species level, was the only significantly enriched taxa when quercetin was supplemented, while 15 species were enriched in rice bran supplementation and 13 were enriched when quercetin and rice bran were supplemented in combination. When comparing the short chain fatty acid production, quercetin supplementation increased isobutyrate production while propionate dominated the quercetin and rice bran combined group. Higher levels of propionate were highly correlated to the lower abundance of the potentially pathogenic family. These findings suggest that the combination of quercetin and rice bran serve to enrich beneficial bacteria and reduce potential opportunistic pathogens. studies are necessary to determine how this synergy of quercetin and rice bran on microbiota impact host health.
PubMed: 34659185
DOI: 10.3389/fmicb.2021.751225 -
Molecular Cell Nov 2021The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of...
The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.
Topics: Acidaminococcus; Animals; Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA; Endodeoxyribonucleases; Endonucleases; Escherichia coli; Gene Editing; Gene Knock-In Techniques; Genetic Techniques; Green Fluorescent Proteins; HEK293 Cells; Humans; In Vitro Techniques; K562 Cells; Mice; Mutagenesis; RNA; Receptors, Chimeric Antigen; Tandem Mass Spectrometry
PubMed: 34648747
DOI: 10.1016/j.molcel.2021.09.021 -
Revista Espanola de Quimioterapia :... Dec 2021
Topics: Abscess; Acidaminococcus; Adult; Female; Fournier Gangrene; Humans; Streptococcus gallolyticus
PubMed: 34645128
DOI: 10.37201/req/070.2021