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allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9(SaCas9)

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Science Bulletin 2019年第21期64卷 1592-1600页

作者： Yajing Liu Jianan Li Changyang Zhou Bin Meng Yu Wei Guang Yang Zongyang Lu Qingmei Shen Yu Zhang Hui Yang Yunbo Qiao School of Life Science and Technology ShanghaiTech UniversityShanghai 201210China Institute of Biochemistry and Cell Biology Chinese Academy of SciencesShanghai 200031China University of Chinese Academy of Sciences Beijing 100049China Institute of Neuroscience State Key Laboratory of NeuroscienceKey Laboratory of Primate NeurobiologyCAS Center for Excellence in Brain Science and Intelligence TechnologyShanghai Institutes for Biological SciencesChinese Academy of SciencesShanghai 200031China Shanghai Institute for Advanced Immunochemical Studies ShanghaiTech UniversityShanghai 200031China Precise Genome Engineering Center School of Life SciencesCuangzhou UniversityGuangzhou 510006China

allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,including Prader-Willi syndrome and cancer.Thus,the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases.To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system,which is an efficient genetargeting technique in various organisms,we examined the targeting efficiency of Staphylococcus aureus Cas9(SaCas9)and Streptococcus pyogenes Cas9(SpCas9)in response to DNA methylation interference.We found that the targeting efficiency of SaCas9,but not SpCas9,was enhanced by targeted DNA demethylation using the d Cas9-Tet1 catalytic domain(CD)but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-d Cas9.An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine(5mC)in a synthesized Cp G-containing context.Further analysis with Ch IP-Q-PCR demonstrated that the non-methylated sequence targeting of Sa Cas9 depends on the binding preference of SaCas9 to non-methylated sequences.Taking advantage of this feature of SaCas9,we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes,H19 and Snrpn,with relatively high efficiencies of 28.6%and 47.4%,respectively.These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation.By using SaCas9,we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.

关键词： allele-specific DNA methylation SaCas9 Imprinting gene

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allele-specific expression and alternative splicing in horse×donkey and cattle×yak hybrids

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Zoological Research 2019年第4期40卷 293-304页

作者： Yu Wang Shan Gao Yue Zhao Wei-Huang Chen Jun-Jie Shao Ni-Ni Wang Ming Li Guang-Xian Zhou Lei Wang Wen-Jing Shen Jing-Tao Xu Wei-Dong Deng Wen Wang Yu-Lin Chen Yu Jiang Key Laboratory of Animal Genetics Breeding and Reproduction of Shaanxi ProvinceCollege of Animal Science and TechnologyNorthwest A&F UniversityYangling Shaanxi 712100China Stake Key Laboratory of Plateau Ecology and Agriculture Qinghai Academy of Animal Science and Veterinary MedicineQinghai UniversityXining Qinghai 810016China State Key Laboratory of Genetic Resources and Evolution Kunming Institute of ZoologyChinese Academy of SciencesKunming Yunnan 650223China Faculty of Animal Science and Technology Yunnan Agricultural UniversityKunming Yunnan 650223China

Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.

关键词： allele-specific alternative splicing allele-specific expression Cis-regulatory elements Hybrid species

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Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification

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Open Journal of Genetics 2011年第2期1卷 13-14页

作者： David J. Bunyan Hilary M. S. Bullman Margaret Lever Sasi D. Saminathan Wee Teik Keng Roziana Araffin David O. Robinson 不详

We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.

关键词： Differential Methylation allele-specific PCR Commercial Kit

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Dynamic Patterns of Gene Expression Additivity and Regulatory Variation throughout Maize Development

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Molecular Plant 2019年第3期12卷 410-425页

作者： Peng Zhou Candice N.Hirsch Steven P.Briggs Nathan M.Springer Department of Plant and Microbial Biology University of MinnesotaSaint PaulMN 55108USA Department of Agronomy and Plant Genetics University of MinnesotaSaint PaulMN 55108USA Division of Biological Sciences University of CaliforniaSanDiegoLaJollaCA92093USA

Gene expression variation is a key component underlying phenotypic variation and heterosis. Transcriptome profiling was performed on 23 different tissues or developmental stages of two maize inbreds, B73 and Mo17, as well as their hybrid. The obtained large-scale datasets provided opportunities to monitor the developmental dynamics of differential expression, additivity for gene expression, and regulatory variation. The transcriptome can be divided into .30 000 genes that are expressed in at least one tissue of one in bred and an additional ~10 000 “silent” genes that are not expressed in any tissue of any genotype, 90% of which are non-syntenic relative to other grasses. Many (.74%) of the expressed genes exhibit differential expression in at least one tissue. However, the majority of genes with differential expression do not exhibit consistent differential expression in different tissues. These genes often exhibit tissue-specific differential expression with equivalent expression in other tissues, and in many cases they switch the directionality of differential expression in different tissues. This suggests widespread variation for tissue-specific regulation of gene expression between the two maize inbreds B73 and Mo17. Nearly 5000 genes are expressed in only one parent in at least one tissue (single parent expression) and 97% of these genes are expressed at mid-parent levels or higher in the hybrid, providing extensive opportunities for hybrid complementation in heterosis. In general, additive expression patterns are much more common than non-additive patterns, and this trend is more pronounced for genes with strong differential expression or single pare nt expressi on. There is relatively little evidence for non-additive expression patterns that are maintained in multiple tissues. The analysis of allele-specific expression allowed classification of cis. and trans-regulatory variation. Genes with c/s-regulatory variation often exhibit additive expression and tend to have more consistent regulatory variation th

关键词： maize gene expression heterosis differential expression non-additive expression allele-specific expression

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A NEW SILENT MUTATION FOUND IN THE CHINESE PAH LOCUS AND ITS ROLE IN THE PRENATAL DIAGNOSIS OF PHENYLKETONURIA

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Science China Chemistry 1991年第5期34卷 606-610页

作者：黄淑帧周霞娣任兆瑞曾溢滔胡流清 Shanghai Institute of Medical Genetics Shanghai Children's Hospital Shanghai 200040 PRC Department of Cell Biology Baylor College of Medicine USA

A silent mutation or sequence polymorphism, A to T substitution at codon 399 in exon11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The fre-quencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09,respectively, based on the analyses of 100 normal individuals and 39 PKU patients usingDNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridizationmethods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagno-sis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPslinkage analysis, was prenatally diagnosed with this genetic marker.

关键词： phenylketonuria phenylalanine hydroxylase (PAH) DNA sequence polymorphism prenatal diagnosis silent mutation polymerase chain reaction (PCR) allele-specific oligonucleotide (ASO) probe Chinese

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Identification of SNPs in barley(Hordeum vulgare L.)by deep sequencing of six reduced representation libraries

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The Crop Journal 2014年第6期2卷 419-425页

作者： Ganggang Guo Dawa Dondup Lisha Zhang Sha Hu Xingmiao Yuan Jing Zhang Key Laboratory of Crop Germplasm Resources and Utilization (MOA) The National Key Facility for Crop Gene Resources and Genetic ImprovementInstitute of Crop Science Chinese Academy of Agricultural Sciences Tibet Academy of Agricultural and Animal Husbandry Sciences

High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) are suitable for accurate genotyping by using the next-generation sequencing(NGS) technology. Reduced representation libraries(RRLs) of five barley accessions and one mutant were sequenced using NGS technology for SNP discovery. Twenty million short reads were generated and the proportion of repetitive sequences was reduced by more than 56%. A total of 6061 SNPs were identified, and 451 were mapped to the draft sequence of the barley genome with pairing reads. Eleven SNPs were validated using length polymorphic allele-specific PCR markers.

关键词： Barley SNP discovery Reduced representation libraries allele-specific PCR

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关于到 colorectal 癌症的 TGFBR1 和危险性的争论

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World Journal of Gastrointestinal Oncology 2012年第1期4卷 1-8页

作者： Laura Valle Hereditary Cancer Program Catalan Institute of Oncology IDIBELL 08908 Hospitalet de Llobregat Spain

Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, ranging from high-penetrance genes to low-risk alleles, account for about 60% of the population-attributable fraction of CRC predisposition. In this context, there is increasing interest in the gene encoding the transforming growth factor β receptor 1 (TGFBR1 ); first when over a decade ago a common polymorphism in exon 1 (rs11466445, TGFBR1 *6A/9A) was suggested to be a risk allele for CRC, then when linkage studies identified the chromosomal region where the gene is located as susceptibility locus for familial CRC, and more recently when the allele-specific expression (ASE) of the gene was proposed as a risk factor for CRC. Published data on the association of TGFBR1 with CRC, regarding polymorphisms and ASE and including sporadic and familial forms of the disease, are often contradictory. This review gives a general overview of the most relevant studies in order to clarify the role of TGFBR1 in the field of CRC genetic susceptibility.

关键词： Transforming growth factor β receptor 1 Transforming growth factor β receptor 1*6A 9q linkage peak allele-specific expression Colorectal cancer risk

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百日咳鲍特菌23S rRNA位点变异AS-PCR检测方法建立

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中国公共卫生 2018年第6期34卷 923-926页

作者：张娟胜李芳栾阳刘莹王增国西安市疾病预防与控制中心陕西710054

目的根据allele-specific PCR(AS-PCR)原理,建立具有良好敏感性与特异性的百日咳鲍特菌23S r RNA位点变异检测方法。方法基于百日咳鲍特菌23S r RNA A2047G位点突变与其对红霉素耐药的相关性,设计包含特异变异位点的引物进行两阶段PCR反... 详细信息

目的根据allele-specific PCR(AS-PCR)原理,建立具有良好敏感性与特异性的百日咳鲍特菌23S r RNA位点变异检测方法。方法基于百日咳鲍特菌23S r RNA A2047G位点突变与其对红霉素耐药的相关性,设计包含特异变异位点的引物进行两阶段PCR反应,根据电泳有无特异大小目的片段的出现,确定百日咳鲍特菌23S r RNA A2047G位点是否有变异。结果以基因序列测定法为金标准的评价结果显示,AS-PCR法检测突变型菌株的灵敏度为96%(144/150),特异度为100%(100/100),kappa=0.95(P<0.01);检测野生型菌株的灵敏度为100%(18/18),特异度为100%(100/100),kappa=1(P<0.01)。结论 AS-PCR方法检测百日咳鲍特菌23S r RNA A2047G位点变异具有较高灵敏度和特异度,适用于普通微生物实验室开展百日咳鲍特菌对红霉素耐药性的快速检测。

关键词：百日咳鲍特菌 allele-specific PCR(AS-PCR) 23S rRNA 红霉素耐药

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Genome-wide screening and analysis of imprinted genes in rapeseed(Brassica napus L.)endosperm

Genome-wide screening and analysis of imprinted genes in rap...

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中国作物学会油料作物专业委员会第八次会员代表大会暨学术...

作者： LIU Jing LI Jun LIU Hong-fang FAN Shi-hang Surinder Singh ZHOU Xue-rong HU Zhi-yong WANG Han-zhong HUA Wei Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences Key Laboratory of Biology and Genetic Improvement of Oil CropsMinistry of Agriculture Agriculture and Food Commonwealth Scientific and Industrial Research Organization

Species-specific genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles.To date,no studies of imprinting have been reported in rapeseed,a tetraploid species.Here,we analyzed global patterns of allelic gene expression in developing rapeseed endosperms from reciprocal crosses between inbred lines YN171 and 93275.14 394 genes were found to harbor diagnostic SNPs between the parental lines.A total of 183 imprinted genes,consisting of 167 maternal expressed genes(MEGs) and 16 paternal expressed genes(PEGs) were identified.Some imprinted genes were validated in different endosperm stages and other parental combinations by RT-PCR analysis.A clear clustering of imprinted genes throughout the rapeseed genome was identified,which was different from most other plants.Methylation analysis to the 104 imprinted genes showed there were 11 genes with differentially methylated regions(DMRs).Unexpectedly,because of lower methylation levels,among the 11 genes,1 gene(BnC04 g0675620) exhibited high CG methylation rate in paternal allele and had big difference between parent alleles.Further research on the imprinting mechanism of BnC04 g0675620 needs to be studied.These results extend our understanding of gene imprinting in plants and provide potential avenues for further research in imprinted genes.

关键词： rapeseed endosperm RNA sequencing imprinting allele-specific DNA methylation

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