Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA from the Streptomyces plasmid pIJ702 was mutagenized in vitro using hydroxylamine and transformed into Streptomyces lividans. One plasmid with temperature-sensitive replication (pMT660) and one plasmid with a temperature-sensitive tyrosinase (pMT661) were isolated. The plasmid pMT661 contains a novel PstI restriction endonuclease site within the tyrosinase gene.
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PMID:Temperature-sensitive mutants of the Streptomyces plasmid pIJ702. 299 43

In two separate studies a BclI-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from Streptomyces antibioticus DNA into two SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using S. lividans 66 as the host. The fragment (1.55 kb) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has two non-essential BclI sites) to generate four new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All six plasmids conferred melanin production (the Mel+ phenotype) on their host. As in the S. antibioticus parent, strains of S. lividans carrying the gene specifying tyrosinase synthesis possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of S. antibioticus; in contrast, the majority remained intracellular in the S. lividans clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in S. antibioticus. Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for BglII, SphI and SstI that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any one of these sites abolished the Mel+ phenotype. The results indicate that pIJ702 is a useful cloning vector with insertional inactivation of the Mel+ character as the basis of clone recognition.
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PMID:Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. 631 61

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish.
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PMID:Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish. 2601 89

Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.
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PMID:Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis. 2626 27

The RNA-guided endonuclease CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is widely used for generating genetically modified mice via zygotic microinjection. Although SpCas9 is a potent mutagen, it requires an NGG proto-spacer adjacent motif (PAM) at the target site, restricting sequence targetability. Here, we show that RNA-guided endonucleases that utilize a range of alternative PAM sequences can edit the mouse genome at the neurog3 (Ngn3) locus: SpCas9 VQR (NGAN PAM), SpCas9 VRER (NGCG), AsCas12a (TTTN), SaCas9 (NNGRRT), and SaCas9 KKH (NNNRRT). Additional experiments targeting tyrosinase and frizzled3 with SaCas9 KKH and its parent protein demonstrated that these endonucleases generated mutations in up to 100% of embryos across three loci. Remarkably, in contrast to wild-type SpCas9, these endonucleases frequently generated mutant embryos that retain unmodified alleles in both template-free and HDR-repair experiments. Our findings broaden PAM recognition options for mouse genome editing and identify SaCas9/SaCas9 KKH as useful alternatives when targeting genes with null lethal phenotypes.
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PMID:Expanding the RNA-Guided Endonuclease Toolkit for Mouse Genome Editing. 3102 Dec 42