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Optimized protoplast isolation and transfection with a breakpoint:accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

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aBIOTECH 2024年第2期5卷 151-168页

作者： Debasmita Panda Subhasis Karmakar Manaswini Dash Swagat Kumar Tripathy Priya Das Sagar Banerjee yiping qi Sanghamitra Samantaray Pradipta Kumar Mohapatra Mirza J.Baig Kutubuddin A.Molla ICAR National Rice Research Institute CuttackOdisha 753006India Department of Botany Ravenshaw UniversityCuttackOdisha 753003India Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic ***-predicted guide RNAs(gRNAs)often fail to induce efficient cleavage at target *** target loci are inaccessible due to complex chromatin ***,there is no suitable tool available to predict the architecture of genomic target sites and their ***,significant time and resources are spent on performing editing experiments with inefficient *** in vitro-cleavage assay could provide a rough assessment of gRNA efficiency,it largely excludes the interference of native genomic *** in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic ***,we developed a modified protocol that offers highly efficient protoplast isolation from rice,Arabidopsis,and chickpea,using a sucrose gradient,transfection using PEG(polyethylene glycol),and validation of single guide RNAs(sgRNAs)cleavage efficiency of *** have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and *** introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea *** dual sgRNAs,our CRISPRdeletion strategy offers straightforward detection of genome editing success by simple agarose gel *** sequencing of PCR products confirmed the editing efficiency of specific ***,we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability,allowing a pause between isolation and *** high-efficiency protocol for protoplast isolation and transfection enables rapid(less than 7 days)validation of sgRNA cleavage efficiency before proceeding with stable *** isolation and transfection method can als

关键词： Protoplast Genome editing Transgene-free edited plants Rice Chickpea

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Versatile plant genome engineering using anti-CRISPR-Cas12a systems

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Science China Life Sciences 2024年

作者： Yao He Shishi Liu Long Chen Dongkai Pu Zhaohui Zhong Tang Xu qiurong Ren Chuan Dong Yawei Wang Danning Wang Xuelian Zheng Fengbiao Guo Tao Zhang yiping qi Yong Zhang Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City Chongqing Key Laboratory of Tree Germplasm Innovation and Utilization School of Life Sciences Southwest University Department of Biotechnology School of Life Sciences and Technology Center for Informational Biology University of Electronic Science and Technology of China Sichuan Institute of Edible Fungi Sichuan Academy of Agricultural Sciences Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Zhongshan Biological Breeding Laboratory/Key Laboratory of Plant Functional Genomics of the Ministry of Education College of AgricultureYangzhou University Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops/Jiangsu Key Laboratory of Crop Genetics and Physiology Yangzhou University Department of Respiratory and Critical Care Medicine Zhongnan Hospital of Wuhan University Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education and School of Pharmaceutical Sciences Wuhan University Department of Plant Science and Landscape Architecture University of Maryland College Park 20742 Institute for Bioscience and Biotechnology Research University of Maryland

CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding. To date, the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants. Here, we conduct in silico analysis to identify putative anti-CRISPR systems for Cas12a. These putative anti-CRISPR proteins, along with known anti-CRISPR proteins, are assessed for their ability to inhibit Cas12a cleavage activity in vivo and in planta. Among all anti-CRISPR proteins tested, AcrVA1 shows robust inhibition of Mb2Cas12a and LbCas12a in E. coli. Further tests show that AcrVA1 inhibits LbCas12a mediated genome editing in rice protoplasts and stable transgenic lines. Impressively, co-expression of AcrVA1 mitigates off-target effects by CRISPR-LbCas12a, as revealed by whole genome sequencing. In addition, transgenic plants expressing AcrVA1 exhibit different levels of inhibition to LbCas12a mediated genome editing, representing a novel way of fine-tuning genome editing efficiency. By controlling temporal and spatial expression of AcrVA1, we show that inducible and tissue specific genome editing can be achieved in plants. Furthermore, we demonstrate that AcrVA1 also inhibits LbCas12a-based CRISPR activation(CRISPRa) and based on this principle we build logic gates to turn on and off target genes in plant cells. Together, we have established an efficient anti-CRISPR-Cas12a system in plants and demonstrate its versatile applications in mitigating off-target effects, finetuning genome editing efficiency, achieving spatial-temporal control of genome editing, and generating synthetic logic gates for controlling target gene expression in plant cells.

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CRISPR ribonucleoprotein-mediated genetic engineering in plants

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Plant Communications 2021年第2期2卷 38-50页

作者： Yingxiao Zhang Brian Iaffaldano yiping qi Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop *** used Agrobacterium-or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target *** efficiency is also highly dependent on the design of the expression cassette and its genomic insertion *** engineering using CRISPR ribonucleoproteins(RNPs)has become an attractive approach with many advantages:DNA/transgene-free editing,minimal off-target effects,and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing *** RNP-mediated genetic engineering has been demonstrated in many plant species,its editing efficiency remains modest,and its application in many species is limited by difficulties in plant regeneration and *** this review,we summarize current developments and challenges in RNPmediated genetic engineering of plants and provide future research directions to broaden the use of this technology.

关键词： CRISPR RNP genetic engineering genome editing transgene free

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CRISPR-Cas nucleases and base editors for plant genome editing

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aBIOTECH 2020年第1期1卷 74-87页

作者： Filiz Gurel Yingxiao Zhang Simon Sretenovic yiping qi Department of Molecular Biology and Genetics Faculty of ScienceIstanbul UniversityVezneciler34134 IstanbulTurkey Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome ***9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living ***12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant ***,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in *** RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,***,genome editing can be achieved by base editors without introducing *** far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall ***,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.

关键词： CRISPR SpCas9 Cas12a Cas12b PAM Cytidine/adenine base editors

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Development of japonica Photo-Sensitive Genic Male Sterile Rice Lines by Editing Carbon Starved Anther Using CRISPR/Cas9

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Journal of Genetics and Genomics 2016年第6期43卷 415-419页

作者： Quanlin Li Dabing Zhang Mingjiao Chen Wanqi Liang Jiaojun Wei yiping qi Zheng Yuan State Key Laboratory of Hybrid Rice Shanghai Jiao Tong Universitye University of Adelaide Joint Centre for Agriculture and HealthSchool of Life Sciences and BiotechnologyShanghai Jiao Tong University Plant Genomics Center School of AgricultureFood and WineUniversity of AdelaideWaite Campus Department of Biology East Carolina University

Rice is one of the most important crops as it supports over25%of total caloric intake for humans（Kusano et al.,2015）.The world population reached 7.3 billion in 2015 and is projected to reach 8.5 billion in 2030（Wo... 详细信息

关键词： arable intake scarce fertility sterile projected sterility breeding crops deletion

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Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

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Plant Communications 2021年第2期2卷 78-88页

作者： Simon Sretenovic Desuo Yin Adam Levav Jeremy D.Selengut Stephen M.Mount yiping qi Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Hubei Academy of Agricultural Sciences Wuhan 430064China Montgomery Blair High School Silver SpringMD 20901USA Center for Bioinformatics and Computational Biology University of MarylandCollege ParkMD 20742USA Department of Cell Biology and Molecular Genetics University of MarylandCollege ParkMD 20742USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

The most popular CRISPR-SpCas9 systemrecognizes canonical NGG protospacer adjacent motifs(PAMs).Previously engineered SpCas9 variants,such as Cas9-NG,favor G-rich PAMs in genome *** this manuscript,we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis,C to T base editing,and A to G base editing at A-rich *** study fills amajor technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants,which greatly expands the targeting scope of ***,our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems,facilitating easy adoption of the systems by *** anticipate that more tools,such as prime editing,homology-directed repair,CRISPR interference,and CRISPR activation,will be further developed based on our promising iSpyMac-Cas9 platform.

关键词： plant genome editing iSpyMacCas9 PAM cytosine base editing adenine base editing

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A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants

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Molecular Plant 2016年第7期9卷 1088-1091页

作者： Tang, Xu Zheng, Xuelian qi, yiping Zhang, Dengwei Cheng, Yan Tang, Aiting Voytas, Daniel F. Zhang, Yong Univ Elect Sci & Technol China Sch Life Sci & Technol Dept Biotechnol Chengdu 610054 Peoples R China Univ Minnesota Dept Genet Cell Biol & Dev Minneapolis MN 55455 USA Univ Minnesota Ctr Genome Engn Minneapolis MN 55455 USA East Carolina Univ Dept Biol Greenville NC 27834 USA

The Streptococcus pyogenes CRISPR-Cas9 system effectively mediates RNA-guided DNA double-strand breaks and is used for genome editing in many different organisms, including plants （Puchta, 2016）. CRISPR-Cas9 is a two-component system in which the Cas9 protein is expressed from a Pol II promoter （Lowder et al., 2015）. In contrast, the sgRNAs are typically expressed from Pol III promoters, such as U6 and U3.

关键词： system multiple heterologous

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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

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Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

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Molecular Plant 2019年第7期12卷 1027-1036页

作者： Zhaohui Zhong Simon Sretenovic qiurong Ren Lijia Yang Yu Bao Caiyan qi Mingzhu Yuan Yao He Shishi Liu Xiaopei Liu Jiaheng Wang Lan Huang Yan Wang Dibin Baby David Wang Tao Zhang yiping qi Yong Zhang Department of Biotechnology School of Life Sciences and Technology Center for Informational Biology University of 曰ectronic Science and Technology of China Room 216 Main Building No. 4 Section 2 North Jianshe Road Chengdu 610054 China Department of Plant Science and Landscape Architecture University of Maryland College Park MD 20742 USA Jiangsu Key Laboratory of Crop Genetics and Physiology Co-Innovation Center for Modern Production Technology of Grain Crops Key Laboratory of Plant Functional Genomics of the Ministry of Education Yangzhou University Yangzhou 225009 China Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China Yangzhou University Yangzhou 225009 China Indian Institute of Science Education and Research Timpati Andhra Pradesh 517507 India Seven Lakes High School Katy TX 77494 USA Institute for Bioscience and Biotechnology Research University of Maryland Rockville MD 20850 USA

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

关键词： xCas9 Cas9-NG high fidelity non-canonical PAM base editing

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Plant Genome Editing Using FnCpfl and LbCpf Nucleases at Redefined and Altered PAM-Sites

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Molecular Plant 2018年第7期11卷 999-1002页

作者： ZhaohuiZhong Yingxiao Zhang qi You XU Tang qiurong Ren Shishi Liu Lijia Yang Yan Wang Xiaopei Liu Binglin Liu Tao Zhang Xuelian Zheng Ysa Le Yong Zhang yiping qi Department of Biotechnology School of Life Sciences and Technology Center for Informational Biology University of Electronic Science andTechnology of China Room 216 Main Building No. 4 Section 2 North J~anshe Road Chengdu 610054 P.R. China Department of Plant Science and Landscape Architecture University of Maryland College Park MD 20742 USA Jiangsu Key Laboratory of Crop Genetics and Physiology Co-InnovationCenter for Modern Production Technology of Grain Crops Key Laboratory ofPlant Functional Genomics of the Ministry of Education Yangzhou University Yangzhou 225009 China Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China Yangzhou University Yangzhou 225009 China Institute for Bioscience and Biotechnology Research University of Maryland Rockville MD 20850 USA These authors contributed equally to this article.

Dear Editor CRISPR from Prevotella and Francisella 1 （Cpfl） is an emerging RNA-guided endonuclease system that relies on thymidine-rich protospacer adjacent motif （PAM） for DNA targeting （Zetsche et al., 2015）. CRISPR-Cpfl has unique features that could be advantageous over the CRISPR-Cas9 system. For example, Cpfl requires only a 42 nt crRNA, while Cas9 uses 100 nt gRNA. While Cas9 generates blunt ends of DNA breaks, the Cpfl cleavage results in 5＇ overhangs distal from the protospacer, which may improve efficiency for NHEJ-based gene insertion. Interestingly, Cpfl proteins also have RNase activity （Fonfara et al., 2016）, which was utilized to process crRNA arrays for multiplexed genome editing in both mammalian systems and plants （Wang et al., 2017; Zetsche et al., 2017）.

关键词： TARGETED MUTAGENESIS CPF1 RICE

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