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RISS 인기검색어
- 검색키워드
- 저자 : Tong Yaojun (검색결과 3 건)
- 무료
- 기관 내 무료
- 유료
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Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST
Tong, Yaojun,Whitford, Christopher M.,Robertsen, Helene L.,Blin, Kai,Jørgensen, Tue S.,Klitgaard, Andreas K.,Gren, Tetiana,Jiang, Xinglin,Weber, Tilmann,Lee, Sang Yup National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.41
<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and common off-target effects remain. To address these concerns, we developed CRISPR-BEST, a DSB-free and high-fidelity single-nucleotide–resolution base editing system for streptomycetes and validated its use by determining editing properties and genome-wide off-target effects. Furthermore, our CRISPR-BEST toolkit supports Csy4-based multiplexing to target multiple genes of interest in parallel. We believe that our CRISPR-BEST approach is a significant improvement over existing genetic manipulation methods to engineer streptomycetes, especially for those strains that cannot be genome-edited using normal DSB-based genome editing systems, such as CRISPR-Cas9.</P><P>Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different <I>Streptomyces</I> species. Particularly in nonmodel actinomycete <I>Streptomyces collinus</I> Tu¨365, CRISPR-cBEST efficiently inactivated the 2 copies of <I>kirN</I> gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.</P>
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Effects of inflow turbulence and slope on turbulent boundary layers over two-dimensional hills
Tong Wang,Shuyang Cao,Yaojun Ge 한국풍공학회 2014 Wind and Structures, An International Journal (WAS Vol.19 No.2
The characteristics of turbulent boundary layers over hilly terrain depend strongly on the hill slope and upstream condition, especially inflow turbulence. Numerical simulations are carried out to investigate the neutrally stratified turbulent boundary layer over two-dimensional hills. Two kinds of hill shape, a steep one with stable separation and a low one without stable separation, and two kinds of inflow condition, laminar and turbulent, are considered. An auxiliary simulation, based on the local differential quadrature method and the recycling technique, is performed to simulate the inflow turbulence to be imposed at the inlet boundary of the simulation with turbulent inflow, which preserves very well in the computational domain. A large separation bubble is established on the leeside of the steep hill with laminar inflow, while the reattachment point moves upstream under turbulent inflow condition. There is stable separation on the lee side of the low hill with laminar inflow, while not with turbulent inflow. Besides increase of turbulence intensity, inflow turbulence can efficiently enhance the speedup around hills.So in practice, it is unreasonable to study wind flow over hilly terrain without considering inflow turbulence.
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Effects of inflow turbulence and slope on turbulent boundary layer over two-dimensional hills
Wang, Tong,Cao, Shuyang,Ge, Yaojun Techno-Press 2014 Wind and Structures, An International Journal (WAS Vol.19 No.2
The characteristics of turbulent boundary layers over hilly terrain depend strongly on the hill slope and upstream condition, especially inflow turbulence. Numerical simulations are carried out to investigate the neutrally stratified turbulent boundary layer over two-dimensional hills. Two kinds of hill shape, a steep one with stable separation and a low one without stable separation, two kinds of inflow condition, laminar turbulent, are considered. An auxiliary simulation, based on the local differential quadrature method and recycling technique, is performed to simulate the inflow turbulence be imposed at inlet boundary of the turbulent inflow, which preserves very well in the computational domain. A large separation bubble is established on the leeside of the steep hill with laminar inflow, while reattachment point moves upstream under turbulent inflow condition. There is stable separation on the side of low hill with laminar inflow, whilw not turbulent inflow. Besides increase of turbulence intensity, inflow can efficiently enhance the speedup around hills. So in practice, it is unreasonable to study wind flow over hilly terrain without considering inflow turbulence.
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