The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement.CRISPR/Cas12i3,which belongs to the ClassⅡTypeⅤ-...The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement.CRISPR/Cas12i3,which belongs to the ClassⅡTypeⅤ-ⅡCas system,has attracted extensive attention recently due to its smaller protein size and less restricted canonical"TTN"protospacer adjacent motif(PAM).However,due to its relatively lower editing efficiency,Cas12i3-mediated multiplex gene editing has not yet been documented in plants.Here,we fused four 5'exonucleases(Exo)including T5E,UL12,PapE,ME15 to the N terminal of an optimized Cas12i3 variant(Cas12i3-5M),respectively,and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines.We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M,respectively.Notably,the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61%across the six tested endogenous genes.We further demonstrated that,although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing,UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three,four,five or six genes with efficiencies of 82.76%,61.36%,52.94%,and 51.06%in rice stable lines,respectively.Together,we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice.The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.展开更多
The effect of the distribution of organic cations CH3NH3^＋（MA^＋） on the stability,electronic structures and optical properties of CH3NH3 Pb I3 perovskite have been investigated using the plane-wave ultrasoft pseuu...The effect of the distribution of organic cations CH3NH3^＋（MA^＋） on the stability,electronic structures and optical properties of CH3NH3 Pb I3 perovskite have been investigated using the plane-wave ultrasoft pseuudopotentials. Generalized gradient approximation and local density approximation are used to optimize the geometries of six models, which are different in the orientation of organic cations. The results show that model C is more stable than others, and the main contribution to the top of valence band is from I 5p states. In the bottom of conduction bands, the main components are Pb 6s states with an overlapping of I 5p states. When the orientation of organic group is transforming, the Pb I6 octahedra will distort and the band structure will alter with it, which affect the generation and migration of photon-generated carriers and optical properties.展开更多
基金partly funded by the Biological Breeding-Major Projects(Grant No.2023ZD04074 to S.L.)the National Natural Science Foundation of China(Grant No.32188102 to L.X.)+2 种基金National Key Research and Development Program of China(Grant No.2021YFF1000204 to L.X)Hainan Seed Industry Laboratory(Grant No.B23CJ0208 to L.X.)the National Engineering Research Centre of Crop Molecular Breeding,and the National Natural Science Foundation of China(Grant No.32100328)。
文摘The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement.CRISPR/Cas12i3,which belongs to the ClassⅡTypeⅤ-ⅡCas system,has attracted extensive attention recently due to its smaller protein size and less restricted canonical"TTN"protospacer adjacent motif(PAM).However,due to its relatively lower editing efficiency,Cas12i3-mediated multiplex gene editing has not yet been documented in plants.Here,we fused four 5'exonucleases(Exo)including T5E,UL12,PapE,ME15 to the N terminal of an optimized Cas12i3 variant(Cas12i3-5M),respectively,and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines.We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M,respectively.Notably,the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61%across the six tested endogenous genes.We further demonstrated that,although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing,UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three,four,five or six genes with efficiencies of 82.76%,61.36%,52.94%,and 51.06%in rice stable lines,respectively.Together,we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice.The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.
基金Supported by the program for National Natural Science Foundation of China(51102150,51472081)Foundation of Hubei University of Technology for High-level Talents(GCRC13014)Development Founds of Hubei Collaborative Innovation Center(HBSKFZD2014003,HBSKFZD2014011,HBSKFZD2015004)
文摘The effect of the distribution of organic cations CH3NH3^＋（MA^＋） on the stability,electronic structures and optical properties of CH3NH3 Pb I3 perovskite have been investigated using the plane-wave ultrasoft pseuudopotentials. Generalized gradient approximation and local density approximation are used to optimize the geometries of six models, which are different in the orientation of organic cations. The results show that model C is more stable than others, and the main contribution to the top of valence band is from I 5p states. In the bottom of conduction bands, the main components are Pb 6s states with an overlapping of I 5p states. When the orientation of organic group is transforming, the Pb I6 octahedra will distort and the band structure will alter with it, which affect the generation and migration of photon-generated carriers and optical properties.
