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Robust Transcriptional Activation in Plants Using Multiplexed crispr-Act2.0 and mTALE-Act Systems

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Molecular Plant 2018年第2期11卷 245-256页

作者： Levi G. Lowder Jianping Zhou Yingxiao Zhang Aimee Malzahn Zhaohui Zhong Tzung-Fu Hsieh Daniel F. Voytas Yong Zhang Yiping Qi Department of Biology East Carolina University Greenville NC 27858 USA Department of Biotechnology School of Life Science and Technology Center for informational Biology University of Electronic Science and Technology of ChinaChengdu 610054 China Department of Plant Science and Landscape Architecture University of Maryland College Park MD 20742 USA Department of Plant and Microbial Biology and Plants for Human Health Institute North Carolina State University North Carolina Research Campus KannapolisNC 28O81 USA Department of Genetics Ceil Biology and Development Center for Genome Engineering University of Minnesota Minneapolis MN 55455 USA Institute for Bioscience and Biotechnology Research University of Maryland Rockville MD 20850 USA

User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated crispr- associated protein 9 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCasg-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 （designated crispr-Act2.0） yielded stronger transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector activation （mTALE-Act） system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than crispr-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex crispr-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.

关键词： crispr gRNA2.0 MS2-VP64 TALE-VP64 multiplex transcriptional activation

crispr technology is revolutionizing the improvement of tomato and other fruit crops

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Horticulture Research 2019年第1期6卷 781-793页

作者： Tian Wang Hongyan Zhang Hongliang Zhu College of Food Science and Nutritional Engineering China Agricultural University100083 BeijingChina Key Laboratory of Food Nutrition and Safety of Shandong Normal University College of Life ScienceShandong Normal University250014 JinanChina

Fruits are major sources of essential nutrients and serve as staple foods in some areas of the *** increasing human population and changes in climate experienced worldwide make it urgent to the production of fruit crops with high yield and enhanced adaptation to the environment,for which conventional breeding is unlikely to meet the ***,clustered regularly interspaced short palindromic repeat(crispr)technology paves the way toward a new horizon for fruit crop improvement and consequently revolutionizes plant *** this review,the mechanism and optimization of the crispr system and its application to fruit crops,including resistance to biotic and abiotic stresses,fruit quality improvement,and domestication are *** and future perspectives are discussed as well.

关键词： crispr crops breeding

An inducible crispr-ON system for controllable gene activation in human pluripotent stem cells

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Protein & Cell 2017年第5期8卷 379-393页

作者： Jianying Guo Dacheng Ma Rujin Huang Jia Ming Min Ye Kehkooi Kee Zhen Xie Jie Na Department of Basic Medical Sciences School of Medicine Center for Stem Cell Biology Tsinghua University Beijing 100084 China MOE Key Laboratory of Bioinformatics and Bioinformatics Division Center for Synthetic and System Biology TNLIST/ Department of Automation Tsinghua University Beijing 100084 China

Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a *** system to efficiently upregulate endogenous genes in hPSCs. A doxycycline （Dox） inducible dCasg-VP64-p65-Rta （dCas9-VPR） transcription activator and a reverse Tet transactivator （rtTA） expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass （ICM） of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

关键词： crispr transcription activation humanpluripotent stem cells NANOG pluripotency

Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for crispr-mediated genome editing

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Journal of Integrative Plant Biology 2014年第4期56卷 343-349页

作者： Yangbin Gao Yunde Zhao Section of Cell and Developmental Biology University of California San Diego9500 Gilman DriveLa Jolla CA 92093-0116 USA

crispr/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by crispr is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/crispr. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.

关键词： CAS9 crispr DNA digestion gRNA genome genomeediting in vitro transcription ribozyme

In vivo delivery of crispr-Cas9 therapeutics:Progress and challenges

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Acta Pharmaceutica Sinica B 2021年第8期11卷 2150-2171页

作者： Matthew Behr Jing Zhou Bing Xu Hongwei Zhang Department of Pharmaceutical Sciences School of Pharmacy–BostonMassachusetts College of Pharmacy and Health SciencesBostonMA 02115USA Department of Endocrinology Tangshan Hospital of Traditional Chinese MedicineTangshan 063000China Department of Chemistry Brandeis UniversityWalthamMA 02454USA

Within less than a decade since its inception,crispr-Cas9-based genome editing has been rapidly advanced to human clinical trials in multiple disease *** it is highly anticipated that this revolutionary technology will bring novel therapeutic modalities to many diseases by precisely manipulating cellular DNA sequences,the low efficiency of in vivo delivery must be enhanced before its therapeutic potential can be fully *** we discuss the most recent progress of in vivo delivery of crispr-Cas9 systems,highlight innovative viral and non-viral delivery technologies,emphasize outstanding delivery challenges,and provide the most updated perspectives.

关键词： crispr Cas9 Genome editing therapy LNP Nanoparticle AAV Lentivirus In vivo

Targeted mutagenesis in Arabidopsis thaliana using crispr-Casi2b/C2ci^°°

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Journal of Integrative Plant Biology 2020年第11期62卷 1653-1658页

作者： Fan Wu Xinyu Qiao Yafei Zhao Ziyi Zhang Yifan Gao Lingfeng Shi Haokun Du Lulu Wang Ya-Jie Zhang Yu Zhang Langyu Liu Quan Wang Dejing Kong School of Biological Science and Engineering Hebei University of Science and TechnologyShijiazhuang 050018China College of Life Sciences Capital Normal UniversityBeijing 100048China Lingnan Laboratory of Modern Agriculture Genome Analysis Laboratory of the Ministry of AgricultureAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518120China State Key Laboratory Breeding Base-Hebei Province Key Laboratory of Molecular Chemistry for Drug Shijiazhuang 050018China

Casi2b/C2ci is a newly identified class 2 crispr endonuclease that was recently engineered for targeted genome editing in mammals and *** explore the potential applications of the crispr-Casi2b system in the dicot Arabidopsis thaliana,we selected BvCasi2b and BhCasi2b v4 for *** successfully used both endonucleases to induce mutations,perform multiplex genome editing,and create large deletions at multiple *** significant mutations were detected at potential off-target *** of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of crisprCasi2b systems for genome editing in Arabidopsis.

关键词： rice. crispr Arabidopsis

Progress and Challenges for Live-cell Imaging of Genomic Loci Using crispr-based Platforms

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Genomics, Proteomics & Bioinformatics 2019年第2期17卷 119-128页

作者： Xiaotian Wu Shiqi Mao Yachen Ying Christopher J.Krueger Antony K. Chen Department of Biomedical Engineering College of Engineering Peking University Beijing 100871 China School of Life Sciences Peking University Beijing 100871 China Wallace H Coulter Department of Biomedical Engineering Georgia Institute of Technology Atlanta GA 30332 USA

Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (crispr)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of crispr-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of crispr-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.

关键词： crispr Cas9 dCas9 Genomic imaging sgRNA

The ScCas9^(++) variant expands the crispr toolbox for genome editing in plants

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Journal of Integrative Plant Biology 2021年第9期63卷 1611-1619页

作者： Taoli Liu Dongchang Zeng Zhiye Zheng Zhansheng Lin Yang Xue Tie Li Xianrong Xie Genglu Ma Yao-Guang Liu Qinlong Zhu State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangzhou 510642China College of Life Sciences South China Agricultural UniversityGuangzhou 510642China Guangdong Laboratory for Lingnan Modern Agriculture Guangzhou 510642China

The development of clustered regularly interspaced palindromic repeats(crispr)-associated protein(Cas) variants with a broader recognition scope is critical for further improvement of crispr/Cas systems. The original Cas9 protein from Streptococcus canis(ScCas9) can recognize simple NNG-protospacer adjacent motif(PAM)targets, and therefore possesses a broader range relative to current crispr/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9^(+) and ScCas9^(++) variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9^(+) and ScCas9^(++) and a nickase variant ScCas9n^(++) to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice(Oryza sativa L.). This analysis revealed that ScCas9^(++) has higher editing efficiency than ScCas9 and ScCas9^(+) in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n^(++) to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n^(++) . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows(C_(-1)-C_(17)) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx(three targets) and OsEui1(two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9^(++) represents a crucial new tool for improving plant editing.

关键词： crispr genome editing base editing ScCas9^(++) rice

Development and Application of crispr/Cas System in Rice

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Rice science 2019年第2期26卷 69-76,I0004-I0006页

作者： REN Jun HU Xixun WANG Kejian WANG Chun State Key Laboratory of Rice Biology China National Rice Research Institute Chinese Academy of Agricultural Sciences

In the past several years, the crispr(clustered regularly interspaced short palindromic repeats)/Cas(crispr-associated protein) system has been harnessed as an efficient and powerful tool for targeted genome editing in different prokaryotic and eukaryotic species. Here, we review the development and application of crispr/Cas system in rice, emphasizing different varieties of crispr/Cas systems have been applied, strategies for multiplex editing, methods for precise gene insertion and replacement, and efficient systems for base editing and site-specific transcriptional regulation. In addition, the biosecurity of crispr/Cas system is also discussed, including transgene-free methods and off-target effects of crispr/Cas system. Thus, the development and application of crispr/Cas system will have significant impact on functional genomic research and variety improvement in rice.

关键词： rice crispr Cas genome editing protospacer adjacent motif