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Ran, Fei

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Ran, Fei

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2014 - 20162

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Author's Publications: search.filters.isAuthorOfPublication.e2d54185-7363-49f3-87c2-e96b5d5c264b

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    CRISPR-Cas: Development and applications for mammalian genome editing
    (2014-06-06) Ran, Fei; Zhang, Feng; Dulac, Catherine; Leschziner, Andres; Camargo, Fernando
    The ability to introduce targeted modifications into genomes and engineer model organisms holds enormous promise for biomedical and technological applications, and has driven the development of tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). To facilitate genome engineering in mammalian cells, we have engineered the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 programmable nuclease systems from Streptococcus pyogenes SF370 (SpCas9) and S. thermophilus LMD-9 (St1Cas9) for mouse and human cell gene editing through heterologous expression of the minimal protein and RNA components. We have demonstrated that Cas9 nucleases can be guided by several short RNAs (sgRNAs) to introduce double stranded breaks (DSB) in the mammalian genome and induce efficient, multiplexed gene modification through non-homologous end-joining-mediated indels or homology-directed repair. Furthermore, we have engineered SpCas9 into a nicking enzyme (SpCas9n) to facilitate recombination while minimizing mutagenic DNA repair processes, and show that SpCas9n can be guided by pairs of appropriately offset sgRNAs to induce DSBs with high efficiency and specificity. In collaboration with Drs. Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, we further report the crystal structure of SpCas9 in complex with the sgRNA and target DNA, and elucidate the structure-function relationship of the ribonucleoprotein complex. Finally, through a metagenomic screen of orthologs, we have identified an additional small Cas9 from Staphylococcus aureus subsp. aureus (SaCas9) that cleaves mammalian endogenous DNA with high efficiency. SaCas9 can be packaged into adeno-associated virus for effective gene modification in vivo. Together, these technologies open up exciting possibilities for applications across basic science, biotechnology, and medicine.
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    Adaptation of CRISPR nucleases for eukaryotic applications
    (Elsevier BV, 2016) Ran, Fei
    Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.