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Truncated gRNA reduces CRISPR/Cas9-mediated off-target rate for MSTN gene knockout in bovines

Journal of Integrative Agriculture 2019年第12期18卷 2835-2843页

作者： ZHOU Zheng-wei CAO Guo-hua LI Zhe HAN Xue-jie LI Chen LU Zhen-yu ZHAO Yu-hang LI Xue-ling state key laboratory of reproductive regulation & breeding of grassland livestocks/Research Center for laboratory Animal Science Inner Mongolia University

The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding. In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of g RNA in reduction of the off-target phenomenon in the bovine MSTN gene knockout fibroblast cell lines was assessed, providing insight into improved methods for livestock breeding. A 20-bp g RNA was designed for the second exon of the bovine MSTN gene, and CRISPR/Cas9-B was constructed to guide the Cas9 protein to the AGAACCAGGAGAAGATGGACTGG site. The alternative CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 vectors were constructed using g RNAs truncated by 1, 2, 3 and 5 bp, respectively. These vectors were then introduced into bovine fetal fibroblasts by the electroporation method, and single cells were obtained by flow cytometry sorting. PCR was performed for each off-target site. All samples were sequenced and analyzed, and finally the efficiency of each vector in target and off-target sites was compared. The CRISPR/Cas9-B vector successfully knocked out the MSTN gene, but the off-target phenomenon was observed. The efficiencies of CRISPR/Cas-B, CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 in triggering gene mutations at MSTN targeting sites were 62.16, 17.39, 7.69, 74.29 and 3.85%, respectively;rates of each at the Off-MSTN-1 locus were 52.86, 0, 0, 8.82 and 0%, respectively;all were 0% at the Off-MSTN-2 locus;rates at the Off-MSTN-3 site were 44.87, 51.72, 86.36, 0 and 50%, respectively. The efficiency of the CRISPR/Cas9-17 plasmid in the MSTN site was higher than that in the CRISPR/Cas9-B plasmid, and the effect at the three off-target sites was significantly lower. This study demonstrated that the CRISPR/Cas9-17 plasmid constructed by truncating 3 bp g RNA can effectively reduce the off-target effect without reducing the efficiency of bovine MSTN gene targeting. This finding will provide more effective gene editing strategy for use

关键词： CRISPR/Cas9 gRNA targeting site off-target rate

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Characterizing Cellular Differentiation Potency and Waddington Landscape via Energy Indicator

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Research 2023年第4期2023卷 619-629页

作者： Hanshuang Li Chunshen Long Yan Hong Liaofu Luo Yongchun Zuo state key laboratory of reproductive regulation and breeding of grassland livestock College of Life SciencesInner Mongolia UniversityHohhot 010070China Department of Physics Inner Mongolia UniversityHohhot 010070China

The precise characterization of cellular differentiation potency remains an open question,which is fundamentally important for deciphering the dynamics mechanism related to cell fate *** quantitatively evaluated the differentiation potency of different stem cells based on the Hopfield neural network(HNN).The results emphasized that cellular differentiation potency can be approximated by Hopfield energy *** then profiled the Waddington energy landscape of embryogenesis and cell reprogramming *** energy landscape at single-cell resolution further confirmed that cell fate decision is progressively specified in a continuous ***,the transition of cells from one steady state to another in embryogenesis and cell reprogramming processes was dynamically simulated on the energy *** two processes can be metaphorized as the motion of descending and ascending ladders,*** further deciphered the dynamics of the gene regulatory network(GRN)for driving cell fate *** study proposes a new energy indicator to quantitatively characterize cellular differentiation potency without prior knowledge,facilitating the further exploration of the potential mechanism of cellular plasticity.

关键词： Hopfield programming landscape

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Whole-genome methylation reveals tissue-specific differences in non-CG methylation in bovine

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Zoological Research 2024年第6期45卷 1371-1384页

作者： Jing Wang Wen Yuan Fang Liu Guang-Bo Liu Xiao-Xiong Geng Chen Li Chen-Chen Zhang Nan Li Xue-Ling Li state key laboratory of reproductive regulation and breeding of grassland livestocks Inner Mongolia UniversityHohhotInner Mongolia 010070China

DNA methylation at non-CG dinucleotides(mCH,H=A,C,T)widely occurs and plays an important role in specific cell types,including pluripotent,neural,and germ ***,the functions and regulatory mechanisms of mCH,particularly in species other than humans and mice,remain inadequately *** this study,we analyzed the distribution of mCH across different bovine tissues,identifying significantly elevated mCH levels in bovine embryonic stem cells(bESCs),as well as brain,spleen,and ileum tissues compared to other *** differences in mCH patterns between somatic cells and bESCs were observed,reflecting distinct base preferences and the differential expression of DNA *** also identified exon methylation in both CG and nonCG contexts,resembling gene-associated methylation patterns observed in *** characterize tissue-specific variations in mCH,we developed a novel method for differential mCH *** indicated that mCH is not randomly distributed but tends to be enriched in tissuespecific functional ***,regression models demonstrated a positional correlation between CG methylation and *** study enhances our understanding of mCH distribution and function in bovine somatic and stem cells,providing new insights into its potential roles across species and *** findings advance knowledge of epigenetic mechanisms,shedding light on the potential involvement of mCH in development and disease processes.

关键词： Epigenetics Non-CG Methylation CHDMRs Comparative Genomics Developmental Biology

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M6A RNA methylation modification and tumor immune microenvironment in lung adenocarcinoma

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Biophysics Reports 2023年第3期9卷 146-158页

作者： Shujuan Li Qianzhong Li Luqiang Zhang Yechen Qi Hui Bai laboratory of Theoretical Biophysics School of Physical Science and Technology Inner Mongolia University The state key laboratory of reproductive regulation and breeding of grassland livestock Inner Mongolia University

Lung adenocarcinoma is one of the deadliest tumors. Studies have shown that N6-methyladenosine RNA methylation regulators, as a dynamic chemical modification, affect the occurrence and development of lung adenocarcinoma. To investigate the relationship between mutations and expression levels of m6A regulators in lung adenocarcinoma, we investigated the mutations and expression levels of38 m6A regulators. We found that mutations in m6A regulatory factors did not affect the changes in expression levels, and 19 differentially expressed genes were identified. All tumor samples were classified into two subtypes based on the expression levels of 19 differentially expressed m6A-regulated genes. Survival analysis showed significant differences in survival between the two subtypes. To explore the relationship between immune cell infiltration and survival in both subtypes, we calculated the infiltration of 23 immune cells in both subtypes, and we found that the subtype with high immune cell infiltration had better survival. We found that subtypes with low tumor purity and high stromal and immune scores had better survival. The m6A-related immune genes were identified by taking the intersection of differentially expressed genes and immune genes in the two isoforms and calculating the Pearson correlation coefficients between the intersecting immune genes and the differentially expressed m6A-regulated genes. Finally, a prognostic model associated with m6A and associated with immunity was developed using prognostic genes screened from m6A-associated immune genes. The predictive power of the model was evaluated and our model was able to achieve good prediction.

关键词： Lung adenocarcinoma M6A RNA methylation regulators Consensus clustering Immune cell infiltrates Tumor immune microenvironment

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The risk model construction of the genes regulated by H3K36me3 and H3K79me2 in breast cancer

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Biophysics Reports 2023年第1期9卷 45-56页

作者： Ling-Yu Wang Lu-Qiang Zhang Qian-Zhong Li Hui Bai laboratory of Theoretical Biophysics School of Physical Science and Technology Inner Mongolia University The state key laboratory of reproductive regulation and breeding of grassland livestock Inner Mongolia University

Abnormal histone modifications(HMs) can promote the occurrence of breast cancer. To elucidate the relationship between HMs and gene expression, we analyzed HM binding patterns and calculated their signal changes between breast tumor cells and normal cells. On this basis, the influences of HM signal changes on the expression changes of breast cancer-related genes were estimated by three different methods. The results showed that H3K79me2 and H3K36me3 may contribute more to gene expression changes. Subsequently, 2109 genes with differential H3K79me2 or H3K36me3 levels during cancerogenesis were identified by the Shannon entropy and submitted to perform functional enrichment analyses. Enrichment analyses displayed that these genes were involved in pathways in cancer, human papillomavirus infection, and viral carcinogenesis. Univariate Cox, LASSO, and multivariate Cox regression analyses were then adopted, and nine potential breast cancer-related driver genes were extracted from the genes with differential H3K79me2/H3K36me3 levels in the TCGA cohort. To facilitate the application, the expression levels of nine driver genes were transformed into a risk score model, and its robustness was tested via time-dependent receiver operating characteristic curves in the TCGA dataset and an independent GEO dataset. At last, the distribution levels of H3K79me2 and H3K36me3 in the nine driver genes were reanalyzed in the two cell lines and the regions with significant signal changes were located.

关键词： Breast cancer Gene expression H3K79me2 H3K36me3 Driver genes

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An increment of diversity method for cell state trajectory inference of time-series scRNA-seq data

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Fundamental Research 2024年第4期4卷 770-776页

作者： Yan Hong Hanshuang Li Chunshen Long Pengfei Liang Jian Zhou Yongchun Zuo state key laboratory of reproductive regulation and breeding of grassland livestock Institutes of Biomedical SciencesCollege of Life SciencesInner Mongolia UniversityHohhot010020China

The increasing emergence of the time-series single-cell RNA sequencing(scRNA-seq)data,inferring developmental trajectory by connecting transcriptome similar cell states(i.e.,cell types or clusters)has become a major *** existing computational methods are designed for individual cells and do not take into account the available time series *** present IDTI based on the Increment of Diversity for Trajectory Inference,which combines time series information and the minimum increment of diversity method to infer cell state trajectory of time-series scRNA-seq *** apply IDTI to simulated and three real diverse tissue development datasets,and compare it with six other commonly used trajectory inference methods in terms of topology similarity and branching *** results have shown that the IDTI method accurately constructs the cell state trajectory without the requirement of starting *** the performance test,we further demonstrate that IDTI has the advantages of high accuracy and strong robustness.

关键词： Increment of diversity Time-series scRNA-seq data Cell state trajectory inference Topology similarity Branching accuracy

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Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

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Journal of Integrative Agriculture 2018年第2期17卷 406-414页

作者： LIU Hui LIU Chang ZHAO Yu-hang HAN Xue-jie ZHOU Zheng-wei WANG Chen LI Rong-feng LI Xue-ling state key laboratory of reproductive regulation & breeding of grassland livestocks Inner Mongolia University Research Center for laboratory Animal Science Inner Mongolia University state key Laboratories of reproductive Medicine Nanjing Medical University Jiangsu key laboratory of Xenotransplantation Nanjing Medical University

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies diff

关键词： myostatin(MSTN) β-casein(CSN2) bovine fetal fibroblasts CRISPR/Cas9 dairy goat fetal fibroblasts eGFP hFat-1 knock-in mutation efficiency TALENs ZFNs

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High-yield production of secoisolariciresinol diglucoside from flaxseed hull by extraction with alcoholic ammonium hydroxide and chromatography on microporous resin

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Food Production, Processing and Nutrition 2021年第1期3卷 450-459页

作者： Cong-Cong Zhuang Chun-Rui Liu Cheng-Bin Shan Zheng Liu Ling Liu Chao-Mei Ma state key laboratory of reproductive regulation&breeding of grassland livestock HohhotChina key laboratory of Herbage&Endemic Crop Biology of Ministry of Education School of Life SciencesInner Mongolia UniversityHohhotP.R.China 010070 Inner Mongolia Yishanyuan Biotechnology Co. LtdHohhotPeople’s Republic of China

This study used alcoholic ammonium hydroxide to directly hydrolyze and extract secoisolariciresinol diglucoside(SDG)from flaxseed hull in a one pot *** optimal extraction conditions,including the concentration of ammonium hydroxide,extraction time,and temperature,were examined in single factor experiments,followed by response surface methodology(RSM)with 3-level,3-factor Box-Behnken *** a result,the optimal extraction conditions were determined as follows:material-liquid ratio 1:20,percentage of reagent ammonium hydroxide(25–28%of NH3 in water)in ethanol 33.7%(pH=12.9),extraction time 4.9 h,and extraction temperature 75.3°*** these conditions,the yield of SDG,as measured by ultra-high-performance liquid chromatography-mass spectrometry,was 23.3 mg/g,consistent with the predicted content of SDG in flaxseed hull(23.0 mg/g).Further,30.0 g of pulverized flaxseed hull was extracted under the optimal conditions,and the extract was subjected to a single run of macroporous resin chromatography to obtain 772.1 mg of a fraction with an SDG content exceeding 76.1%.Subsequent chromatography on Sephadex LH20,yielded 602.8 mg SDG of 98.0%purity,and the yield was 20.1 mg/g(2.0%)from flaxseed ***,one-pot hydrolysis and extraction of SDG using alcoholic ammonium hydroxide is simple,and of high-yield.

关键词： Flaxseed hull SDG Extraction Alkaline hydrolysis RSM

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Study on the spatial distribution patterns of histone modifications in Hippo pathway genes

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Biophysics Reports 2021年第1期7卷 71-79页

作者： Wenxia Su Dimeng Zhang Qiang Zhang Qianzhong Li College of Science Inner Mongolia Agriculture University laboratory of Theoretical Biophysics School of Physical Science and Technology Inner Mongolia University The state key laboratory of reproductive regulation and breeding of grassland livestock Inner Mongolia University

Hippo pathway can regulate cell division, differentiation and apoptosis, and control the shape and size of organs. To study the distribution patterns of histone modifications of Hippo pathway genes in embryonic stem cells is helpful to understand the molecular regulation mechanism of histone modification and Hippo pathway on stem cell self-renewal. In this study, 19 genes of Hippo pathway including YAP, TAZ, LATS1/2, MST1 and SAV1, and eight histone modifications in embryonic stem cells were chosen to study the spatial distribution patterns of histone modifications. It was found that there were obvious type specificity and the location preference of target regions in the distributions of histone modifications, and H3 K4 me3 and H3 K36 me3 played the most important regulatory roles. Through the correlation analysis of histone modifications, a histone modification functional cluster composed of H3 K4 ac, H3 K4 me3, H3 K9 ac and H3 K27 ac was detected in YAP. In addition, the spatial distribution patterns of histone modifications in Hippo pathway genes were obtained, which provided a new theoretical reference for elucidating the mechanism of histone modifications regulating the gene expression of Hippo pathway, and for revealing the molecular regulatory mechanism of histone modifications affecting the self-renewal of embryonic stem cells by regulating the Hippo pathway.

关键词： Embryonic stem cell Hippo pathway Histone modification Spatial distribution pattern

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A prognostic model for acute myeloid leukemia based on ferroptosis-related lncRNA and immune infiltration analysis

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Biophysics Reports 2024年第6期10卷 377-387页

作者： Shuhan Liu Yingli Chen Qianzhong Li Zhiyu Fan Menglan Li Pengyu Du laboratory of Theoretical Biophysics School of Physical Science and Technology Inner Mongolia University The state key laboratory of reproductive regulation and breeding of grassland livestock Inner Mongolia University

Acute myeloid leukemia(AML) is a rare tumor that invades the blood and bone marrow, it is rapidly progressive, highly aggressive, and difficult to cure. Studies have shown that long non-coding RNA(lncRNA) and ferroptosis play important roles in AML. However, few studies have been done on ferroptosis-related lncRNA for AML. To investigate the role of ferroptosis-related lncRNA in AML prognosis, we screened the differentially expressed genes related to ferroptosis and lncRNA. Ferroptosisrelated lncRNA associated with AML prognosis was obtained by Pearson correlation analysis. By using univariate Cox analysis, least absolute shrinkage and selection operator(LASSO) analysis, and multivariate Cox analysis, the ten prognostic genes were used for constructing the prognostic model. The model was then validated using a Kaplan-Meier analysis and Cox regression analysis. The ROC results have shown that the model could better predict AML survival. We identified some mutated genes that may affect the poor prognosis based on the somatic mutation analysis. The enrichment pathway analysis of prognostic genes revealed that these genes were mainly enriched in some immune pathways and cancer pathways. By immune infiltration analysis, we found that high-risk patients may respond better to immunotherapy.

关键词： Acute myeloid leukemia Ferroptosis Long non-coding RNA Prognosis Immune infiltrates

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