Human knock out Pd-1 gene sequence
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens (human)
SUBMITTER:
PROVIDER: S-BSST1619 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description: Not available
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Project description:Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.
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Project description:The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.
| S-EPMC5762654 | biostudies-literature
Project description:Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.
| S-EPMC6722079 | biostudies-literature
Project description:There is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.
| S-EPMC8800938 | biostudies-literature
Project description:We will perform RNAseq to evaluate the effects of the loss of a list of TSGs on the transcriptome.
| EGAS00001002262 | EGA
Project description:This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.
| S-EPMC8163928 | biostudies-literature
Project description:Recent genome-wide association studies of age-at-onset in Huntington's disease (HD) point to distinct modes of potential disease modification: altering the rate of somatic expansion of the HTT CAG repeat or altering the resulting CAG threshold length-triggered toxicity process. Here, we evaluated the mouse orthologs of two HD age-at-onset modifier genes, FAN1 and RRM2B, for an influence on somatic instability of the expanded CAG repeat in Htt CAG knock-in mice. Fan1 knock-out increased somatic expansion of Htt CAG repeats, in the juvenile- and the adult-onset HD ranges, whereas knock-out of Rrm2b did not greatly alter somatic Htt CAG repeat instability. Simultaneous knock-out of Mlh1, the ortholog of a third HD age-at-onset modifier gene (MLH1), which suppresses somatic expansion of the Htt knock-in CAG repeat, blocked the Fan1 knock-out-induced acceleration of somatic CAG expansion. This genetic interaction indicates that functional MLH1 is required for the CAG repeat destabilizing effect of FAN1 loss. Thus, in HD, it is uncertain whether the RRM2B modifier effect on timing of onset may be due to a DNA instability mechanism. In contrast, the FAN1 modifier effects reveal that functional FAN1 acts to suppress somatic CAG repeat expansion, likely in genetic interaction with other DNA instability modifiers whose combined effects can hasten or delay onset and other CAG repeat length-driven phenotypes.
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Project description:A novel line of mutant mice [monoamine oxidase A knockout (MAOA KO)] harboring a spontaneous point nonsense mutation in exon 8 of the MAO A gene was serendipitously identified in a 129/SvEvTac colony. This mutation is analogous to the cause of a rare human disorder, Brunner syndrome, characterized by complete MAO A deficiency and impulsive aggressiveness. Concurrent with previous studies of MAO A KO mice generated by insertional mutagenesis ('Tg8'), MAOA(A863T) KO lack MAO A enzyme activity and display enhanced aggression toward intruder mice. MAOA(A863T) KO, however, exhibited lower locomotor activity in a novel, inescapable open field and similar immobility during tail suspension compared with wild type, observations which differ from reports of Tg8. These findings consolidate evidence linking MAO A to aggression and highlight subtle yet distinctive phenotypical characteristics.
| S-EPMC3435113 | biostudies-literature
Project description:Chlamydomonas reinhardtii is being transformed from a model organism to an industrial organism for the production of pigments, fatty acids, and pharmaceuticals. Genetic modification has been used to increase the economic value of C. reinhardtii. However, low gene-editing efficiency and position-effects hinder the genetic improvement of this microorganism. Recently, site-specific double-stranded DNA cleavage using CRISPR-Cas9 system has been applied to regulate a metabolic pathway in C. reinhardtii. In this study, we proved that site-specific gene expression can be induced by CRISPR-Cas9-mediated double-strand cleavage and non-homologous end joining (NHEJ) mechanism. The CRISPR-Cas9-mediated knock-in method was adopted to improve gene-editing efficiency and express the reporter gene on the intended site. Knock-in was performed using a combination of ribonucleoprotein (RNP) complex and DNA fragment (antibiotics resistance gene). Gene-editing efficiency was improved via optimization of a component of RNP complex. We found that when the gene CrFTSY was targeted, the efficiency of obtaining the desired mutant by the knock-in method combined with antibiotic resistance was nearly 37%; 2.5 times higher than the previous reports. Additionally, insertion of a long DNA fragment (3.2 and 6.4 kb) and site-specific gene expression were analyzed. We demonstrated the knock-out phenotype of CrFTSY and on-site inserted gene expression of luciferase and mVenus at the same time. This result showed that CRISPR-Cas9-mediated knock-in can be used to express the gene of interest avoiding position-effects in C. reinhardtii. This report could provide a new perspective to the use of gene-editing. Furthermore, the technical improvements in genetic modification may accelerate the commercialization of C. reinhardtii.
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