CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deleti...CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.展开更多
Jute(Corchorus spp.)is a member of the Malvaceae family,which comprises more than 100 species.The systematic positions of jute species have remained unsettled.Chloroplasts are maternally inherited and their genomes ar...Jute(Corchorus spp.)is a member of the Malvaceae family,which comprises more than 100 species.The systematic positions of jute species have remained unsettled.Chloroplasts are maternally inherited and their genomes are widely used for plant phylogenetic studies.In the present study,the chloroplast genomes of Corchorus capsularis and C.olitorius were assembled,with sizes of respectively 161,088 and 161,766 bp.Both genomes contained 112 unique genes(78 protein-coding,four rRNA,and 30 tRNA genes).Four regionswith high variation between the two species were located in single-copy rather than inverted-repeat regions.A total of 66 simple sequence repeats(SSRs)were identified in the C.capsularis chloroplast genome and 56 in that of C.olitorius.Comparison of the two chloroplast genome sequences permitted the evaluation of nucleotide variation including 2417 single-nucleotide polymorphisms sites and 294 insertion or deletion sites,of which one marker(cpInDel 205)could discriminate the two jute species.Comparison of the C.capsularis and C.olitorius chloroplast genomeswith those of other species in theMalvaceae revealed breakpoints in the accD locus,which is involved in fatty acid synthesis,in C.capsularis and C.olitorius.This finding suggests that genes from the chloroplast genomemight have been transferred to the nuclear genome in some Corchorus species.This hypothesis was supported by synteny analysis of the accD region among the nuclear,chloroplast,and mitochondrial genomes.To our knowledge,this is the first report of the assembled chloroplast genome sequences of C.capsularis and C.olitorius.C.capsularis and C.olitorius are closely related to Gossypium species and there are abundant microstructure variations between these two genera.These results will expand our understanding of the systematics of species in the Malvaceae.展开更多
目的探讨胎儿先天性心脏痫基因型与临床表型之间的关联性，旨在为先天性心脏病遗传学咨询发病机制研究提供信息。方法选取2012年9月至2015年6月间首都医科大学北京安贞医院胎儿心脏病母胎医学会诊中心胎儿心脏畸形数据库心肌等组织标本6...目的探讨胎儿先天性心脏痫基因型与临床表型之间的关联性，旨在为先天性心脏病遗传学咨询发病机制研究提供信息。方法选取2012年9月至2015年6月间首都医科大学北京安贞医院胎儿心脏病母胎医学会诊中心胎儿心脏畸形数据库心肌等组织标本62例，按区段月不胎学分类法进行分型，并送深圳华大基因研究院利用全基因组低覆盖度测序检测染色体缺失／重复，目标区域捕获测序检测先天性心脏病相关基因的单核苷酸变异及小的捅入缺失片段。结果（1）本研究中胎儿先天性心脏病以圆锥动脉干畸形（conotruncal defects，CTD）最常见（69．4％，43／62）。（2）共30例胎儿检出〉100kb的拷贝数变异（copy number variations，CNVs）（48．4％，30／62），其中11例检出〉1Mb的CNVs，11例中7例为致病意义明确，7例中有6例心脏临床表刊累及CTD。（3）5例胎儿检出已知及疑似致病基因，其中4例胎儿存在心室流出道梗阻。结论本研究中胎儿的心脏临床表型以CTD常见，检出致病意义明确的胎儿心脏表型亦以CTD常见；检出疑似致病突变基因的胎儿，其临床表型以心事流出道梗阻多见。展开更多
Based on the gene-protein-reaction (GPR) model of S. cerevisiae_iND750 and the method of constraint-based analysis, we first calculated the metabolic flux distribution of S. cere-visiae_iND750. Then we calculated the ...Based on the gene-protein-reaction (GPR) model of S. cerevisiae_iND750 and the method of constraint-based analysis, we first calculated the metabolic flux distribution of S. cere-visiae_iND750. Then we calculated the deletion impact of 438 calculable genes, one by one, on the metabolic flux redistribution of S. cere-visiae_iND750. Next we analyzed the correlation between v (describing deletion impact of one gene) and d (connection degree of one gene) and the correlation between v and Vgene (flux sum controlled by one gene), and found that both of them were not of linear relation. Furthermore, we sought out 38 important genes that most greatly affected the metabolic flux distribution, and determined their functional subsystems. We also found that many of these key genes were related to many but not several subsystems. Because the in silico model of S. cere-visiae_iND750 has been tested by many ex-periments, thus is credible, we can conclude that the result we obtained has biological sig-nificance.展开更多
The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Com...The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.展开更多
We have isolated a recessive rice mutant, designated as indeterminate growth （ing）, which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms. Rough mapping ...We have isolated a recessive rice mutant, designated as indeterminate growth （ing）, which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms. Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion, which corresponds to a 103 kb region in the Nipponbare genome, containing nine annotated genes on chromosome 3. Of these annotated genes, the SLR1 gene encoding a DELLA protein is the only one that is well characterized in its function, and its null mutation, which is caused by a single base deletion in the middle of the intronless SLR1 gene, confers a slender phenotype that bears close resemblance to the ing mutant phenotype. The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene, and the ing mutant appears to be the first characterized mutant having the entire SLR1 sequence deleted. Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene, whose dysfunction must result in a lethal phenotype.展开更多
基金supported by grants from the National Transgenic Science and Technology Program of China(2019ZX08010-003,2019ZX08010-001,2018ZX0801002B)the National Key Research and Development Program of China(2016YFD0100602)+1 种基金the Scientific Program of Beijing Municipal Commission of Science and Technology(Z171100001517001)the National Natural Science Foundation of China(31672015)to J.L.Q。
文摘CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.
基金supported by the National Natural Science Foundation of China(31771369)the China Agriculture Research System for Crops of Bast and Leaf Fiber,China(nycytx-19-E06)。
文摘Jute(Corchorus spp.)is a member of the Malvaceae family,which comprises more than 100 species.The systematic positions of jute species have remained unsettled.Chloroplasts are maternally inherited and their genomes are widely used for plant phylogenetic studies.In the present study,the chloroplast genomes of Corchorus capsularis and C.olitorius were assembled,with sizes of respectively 161,088 and 161,766 bp.Both genomes contained 112 unique genes(78 protein-coding,four rRNA,and 30 tRNA genes).Four regionswith high variation between the two species were located in single-copy rather than inverted-repeat regions.A total of 66 simple sequence repeats(SSRs)were identified in the C.capsularis chloroplast genome and 56 in that of C.olitorius.Comparison of the two chloroplast genome sequences permitted the evaluation of nucleotide variation including 2417 single-nucleotide polymorphisms sites and 294 insertion or deletion sites,of which one marker(cpInDel 205)could discriminate the two jute species.Comparison of the C.capsularis and C.olitorius chloroplast genomeswith those of other species in theMalvaceae revealed breakpoints in the accD locus,which is involved in fatty acid synthesis,in C.capsularis and C.olitorius.This finding suggests that genes from the chloroplast genomemight have been transferred to the nuclear genome in some Corchorus species.This hypothesis was supported by synteny analysis of the accD region among the nuclear,chloroplast,and mitochondrial genomes.To our knowledge,this is the first report of the assembled chloroplast genome sequences of C.capsularis and C.olitorius.C.capsularis and C.olitorius are closely related to Gossypium species and there are abundant microstructure variations between these two genera.These results will expand our understanding of the systematics of species in the Malvaceae.
文摘目的探讨胎儿先天性心脏痫基因型与临床表型之间的关联性，旨在为先天性心脏病遗传学咨询发病机制研究提供信息。方法选取2012年9月至2015年6月间首都医科大学北京安贞医院胎儿心脏病母胎医学会诊中心胎儿心脏畸形数据库心肌等组织标本62例，按区段月不胎学分类法进行分型，并送深圳华大基因研究院利用全基因组低覆盖度测序检测染色体缺失／重复，目标区域捕获测序检测先天性心脏病相关基因的单核苷酸变异及小的捅入缺失片段。结果（1）本研究中胎儿先天性心脏病以圆锥动脉干畸形（conotruncal defects，CTD）最常见（69．4％，43／62）。（2）共30例胎儿检出〉100kb的拷贝数变异（copy number variations，CNVs）（48．4％，30／62），其中11例检出〉1Mb的CNVs，11例中7例为致病意义明确，7例中有6例心脏临床表刊累及CTD。（3）5例胎儿检出已知及疑似致病基因，其中4例胎儿存在心室流出道梗阻。结论本研究中胎儿的心脏临床表型以CTD常见，检出致病意义明确的胎儿心脏表型亦以CTD常见；检出疑似致病突变基因的胎儿，其临床表型以心事流出道梗阻多见。
文摘Based on the gene-protein-reaction (GPR) model of S. cerevisiae_iND750 and the method of constraint-based analysis, we first calculated the metabolic flux distribution of S. cere-visiae_iND750. Then we calculated the deletion impact of 438 calculable genes, one by one, on the metabolic flux redistribution of S. cere-visiae_iND750. Next we analyzed the correlation between v (describing deletion impact of one gene) and d (connection degree of one gene) and the correlation between v and Vgene (flux sum controlled by one gene), and found that both of them were not of linear relation. Furthermore, we sought out 38 important genes that most greatly affected the metabolic flux distribution, and determined their functional subsystems. We also found that many of these key genes were related to many but not several subsystems. Because the in silico model of S. cere-visiae_iND750 has been tested by many ex-periments, thus is credible, we can conclude that the result we obtained has biological sig-nificance.
基金supported by the National Natural Sci-ence Foundation of China (30671563,30700597)the Natural Science Foundation of Gansu Province,China (0803RJZA050)
文摘The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.
基金supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences(PROBRAIN to S.I.) from Bio-oriented Technology Research Advancement Institution(BRAIN) in Japangrants from the Ministry of Education,Culture,Sports, Science,and Technology of Japan(No.17207002 to S.I.and 22780007 to K.T.)+1 种基金supported by the Global COE Program(to H.K.and S.I.)the NIBB Cooperative Research Program(9-151 to H.K.and 9-153 to M.M.),and the Graduate University for Advanced Studies(Sokendai)
文摘We have isolated a recessive rice mutant, designated as indeterminate growth （ing）, which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms. Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion, which corresponds to a 103 kb region in the Nipponbare genome, containing nine annotated genes on chromosome 3. Of these annotated genes, the SLR1 gene encoding a DELLA protein is the only one that is well characterized in its function, and its null mutation, which is caused by a single base deletion in the middle of the intronless SLR1 gene, confers a slender phenotype that bears close resemblance to the ing mutant phenotype. The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene, and the ing mutant appears to be the first characterized mutant having the entire SLR1 sequence deleted. Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene, whose dysfunction must result in a lethal phenotype.
