The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still chal...The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.展开更多
What is the most favorite and original chemistry developed in your research group?The visible light photosensitizer DPZ.How did you get into this specific field?Could you please share some experiences with our readers...What is the most favorite and original chemistry developed in your research group?The visible light photosensitizer DPZ.How did you get into this specific field?Could you please share some experiences with our readers?Since I began my postdoctoral work at NUS,chiral hydrogen-bonding catalysis,an important branch of asymmetric organocatalysis,has become the general area of focus of my research.To broaden the applications of this strategy,I proposed to exploit photocatalysis to generate highly reactive radical intermediates,thus overcoming the current limitations in both reaction and substrate types stemming from the low energy of hydrogen-bonding interactions.The development of highly efficient organophotocatalysts and explorations of transition metal-free cooperative photocatalysis and chiral hydrogen-bonding catalysis have therefore been my research focus since 2013.My students and I strive to follow the saying"stick to the research direction,thoroughly understand the scientific challenges,and face those challenges with optimism and determination".展开更多
An enantioselective three-component propargyloxylation reaction of propargyl alcohols,pyridotriazoles,and imines has been realized by cooperative catalysis with dirhodium complex and chiral phosphoric acid under mild ...An enantioselective three-component propargyloxylation reaction of propargyl alcohols,pyridotriazoles,and imines has been realized by cooperative catalysis with dirhodium complex and chiral phosphoric acid under mild conditions.This is the first example of a catalytic asymmetric three-component propargyloxylation reaction,which provides practical access to chiral polyfunctionalized succinate derivatives with adjacent quaternary and tertiary stereocenters in good to high yields with excellent enantioselectivity.In addition to the alkyne motif,pyridyl,alkoxy,amino,and alkenyl species are all tolerated under the reaction conditions.Notably,the utility of the current method is demonstrated by catalytic cyclization of the product alkyne into a variety of heterocyclic structures without loss of enantiomeric purity.展开更多
基金This work was supported by Beijing Academy of Agriculture&Forestry Scien ces,Beijing,ChinaThe funding in eluded Innovative Team Con-struction Project of BAAFS(JNKYT201603)the Beijing Scholars Program(BSP041).
文摘The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.
基金We sincerely appreciate all former and present members ofour group and the many collaborators and friends who havehelped,whose names are listed in part in the relevant references.We also thank generous financial support from the National Nat-ural Science Foundation of China(Nos.21072044,21672052 and21925103)NCET-11-0938,Henan Province and Henan University.
文摘What is the most favorite and original chemistry developed in your research group?The visible light photosensitizer DPZ.How did you get into this specific field?Could you please share some experiences with our readers?Since I began my postdoctoral work at NUS,chiral hydrogen-bonding catalysis,an important branch of asymmetric organocatalysis,has become the general area of focus of my research.To broaden the applications of this strategy,I proposed to exploit photocatalysis to generate highly reactive radical intermediates,thus overcoming the current limitations in both reaction and substrate types stemming from the low energy of hydrogen-bonding interactions.The development of highly efficient organophotocatalysts and explorations of transition metal-free cooperative photocatalysis and chiral hydrogen-bonding catalysis have therefore been my research focus since 2013.My students and I strive to follow the saying"stick to the research direction,thoroughly understand the scientific challenges,and face those challenges with optimism and determination".
基金supported by the National Natural Science Foundation of China(nos.21971262 and 81973176)the Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery(no.2019B030301005)+1 种基金the National Mega-Project for Innovative Drugs(no.2019ZX09721001-006-001)the Program for Guangdong Introducing Innovative and Entrepreneurial Teams(no.2016ZT06Y337).
文摘An enantioselective three-component propargyloxylation reaction of propargyl alcohols,pyridotriazoles,and imines has been realized by cooperative catalysis with dirhodium complex and chiral phosphoric acid under mild conditions.This is the first example of a catalytic asymmetric three-component propargyloxylation reaction,which provides practical access to chiral polyfunctionalized succinate derivatives with adjacent quaternary and tertiary stereocenters in good to high yields with excellent enantioselectivity.In addition to the alkyne motif,pyridyl,alkoxy,amino,and alkenyl species are all tolerated under the reaction conditions.Notably,the utility of the current method is demonstrated by catalytic cyclization of the product alkyne into a variety of heterocyclic structures without loss of enantiomeric purity.
