Publications by authors named "Rongfang Xu"

19 Publications

  • Page 1 of 1

Development of an efficient plant dual cytosine and adenine editor.

J Integr Plant Biol 2021 Jun 30. Epub 2021 Jun 30.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, China.

An enhanced CDA-like (eCDAL) was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editor version 1(pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.
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http://dx.doi.org/10.1111/jipb.13146DOI Listing
June 2021

Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice.

Nat Plants 2021 Jul 10;7(7):888-892. Epub 2021 Jun 10.

Key Laboratory of Rice Genetics & Breeding, Institute of Rice Research, Anhui Academy of Agricultural Science, Hefei, China.

Base-editing-library-induced high density nucleotide substitutions have been applied to screen functional mutations in plants. However, due to limitations in the scope and conversion specificity of base editors, many desired mutations at pivotal protein sites may be overlooked. Here, we developed a prime-editing-library-mediated saturation mutagenesis (PLSM) method to substantially increase the diversity of amino acid substitutions at target sites for in planta screening. At six conserved residues of OsACC1, 16 types of herbicide-resistance-endowing mutations were identified. Most of these mutations exhibit reliable tolerance to aryloxyphenoxypropionate herbicides and have not been reported or applied in rice breeding. In addition, the advantage of PLSM was further shown by comparing the base-editing-mediated mutagenesis at the selected targets. The PLSM method established in this study has great potential for the direct evolution of genes related to agronomically important traits for crop improvement.
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http://dx.doi.org/10.1038/s41477-021-00942-wDOI Listing
July 2021

Real-world comparison of direct-acting oral anticoagulants and vitamin K antagonists in chronic kidney disease: a systematic review and meta-analysis.

Expert Rev Hematol 2021 May 5;14(5):493-502. Epub 2021 May 5.

Department of Nephrology, Changxing People's Hospital, Huzhou, Zhejiang Province, P.R. China.

: No clear clinical guidelines exist on anticoagulant use for chronic kidney disease (CKD) patients. We compared the efficacy and safety of direct-acting oral anticoagulants (DOACs) vs. vitamin K antagonists (VKA) in patients with CKD by pooling data from real-world observational studies.: This systematic review searched PubMed, Embase, and CENTRAL databases and pooled multivariable-adjusted hazard ratios (HR) of outcomes.: Fifteen studies were included. Our results indicated a small but significant reduction in the risk of all-cause mortality (p = 0.01), stroke or systemic embolism (p = 0.03), and major bleeding (p = 0.01) with DOAC as compared to VKA. In subgroup analysis based on the severity of CKD, no difference in the risk of stroke or systemic embolism was noted in any subgroups. The risk of mortality was reduced only in patients with moderate-severe or severe CKD and the risk of major bleeding was reduced only in patients with moderate-severe or moderate CKD.: DOACs are associated with only a modest reduction in stroke or systemic embolism, major bleeding, and mortality when compared to VKA in CKD patients. Reduction in mortality and major bleeding with DOAC may only be seen in moderate-to-severe CKD patients.
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http://dx.doi.org/10.1080/17474086.2021.1920012DOI Listing
May 2021

Effects of graphene oxide on tomato growth in different stages.

Plant Physiol Biochem 2021 May 12;162:447-455. Epub 2021 Mar 12.

School of Life Sciences, Shanxi Datong University, Datong, 037009, China.

The nano-carbon graphene has unique structural and physicochemical properties, which are conducive to various biomedical applications. We assessed the effect of graphene oxide (GO) on tomato plants at the seedling and mature stages in terms of morphological and biochemical indices. GO treatment significantly improved the shoot/stem growth of tomato in a dose-dependent manner by increasing the cortical cells number, cross-sectional area, diameter and vascular-column area. In addition, GO also promoted the morphological development of the root system and increased biomass accumulation. The surface area of root tips and hairs of tomato plants treated with 50 mg/L and 100 mg/L GO were significantly greater compared to the untreated control. At the molecular level, GO induced the expression of root development-related genes (SlExt1 and LeCTR1) and inhibited the auxin-responsive gene (SlIAA3). However, 50 mg/L and 100 mg/L GO significantly increased the root auxin content, which in turn increased the number of fruits and hastened fruit ripening compared to the control plants. Taken together, GO can improve the tomato growth when used at the appropriate concentration, and is a promising nano-carbon material for agricultural use.
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http://dx.doi.org/10.1016/j.plaphy.2021.03.013DOI Listing
May 2021

Preparation of a CaTiO/Al/Pr/Sm nanocomposite for enrichment of exosomes in human serum.

Talanta 2021 May 5;226:122186. Epub 2021 Feb 5.

The National & Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha, 410081, PR China; State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences, Hunan Normal University, Changsha, 410081, PR China. Electronic address:

Exosomes (30-200 nm) play important roles in intercellular communication. Because their contents differ between healthy individuals and subjects diagnosed with various diseases, exosomes have been regarded as potential sources of biomarkers for clinical diagnosis. However, the accuracy of diagnosis by exosomal biomarkers is highly dependent on the extraction efficiency, yield, and the quality of exosomes. Hence, inexpensive, convenient, and fast exosome separation methods are required. In the present study, the CaTiO/Al/Pr/Sm nanocomposite was synthesized and applied in highly selective and efficient separation of exosomes. Notably, the developed material exhibited higher specificity and efficiency than commercially available TiO. Moreover, CaTiO/Al/Pr/Sm could be reused at least three times without any significant decrease in efficiency. The synthesized material was also used for the extraction of exosomes from the serums of patients with Alzheimer's disease (AD) and healthy controls. The exosomes were subjected to two-dimensional gel electrophoresis (2-DE) separation and matrix-assisted laser desorption/ionization-time of flight (MALDI TOF/TOF) mass spectrometry analysis. It was found that five proteins in the exosomes were evidently upregulated, while one protein was downregulated. Among the detected proteins, serum amyloid P-component (SAP) has been reported to be closely related to pathogenesis of AD. The obtained results indicated that the developed method involving separation and analysis of serum exosomes could be used for disease diagnosis or postoperative clinical monitoring.
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http://dx.doi.org/10.1016/j.talanta.2021.122186DOI Listing
May 2021

Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.

Mol Plant 2021 02 28;14(2):352-360. Epub 2020 Dec 28.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, P.R. China. Electronic address:

Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.
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http://dx.doi.org/10.1016/j.molp.2020.12.017DOI Listing
February 2021

Development of Plant Prime-Editing Systems for Precise Genome Editing.

Plant Commun 2020 May 8;1(3):100043. Epub 2020 Apr 8.

Key Laboratory of Rice Genetics & Breeding, Institute of Rice Research, Anhui Academy of Agricultural Science, Hefei 230031, China.

Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T plants, pPE2-generated mutants occurred with 0%-31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.
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http://dx.doi.org/10.1016/j.xplc.2020.100043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747961PMC
May 2020

SpCas9-NG self-targets the sgRNA sequence in plant genome editing.

Nat Plants 2020 03 24;6(3):197-201. Epub 2020 Feb 24.

Key Laboratory of Rice Genetics & Breeding, Institute of Rice Research, Anhui Academy of Agricultural Science, Hefei, China.

Streptococcus pyogenes Cas9 (SpCas9)-NG recognizes NGN protospacer adjacent motifs and expands the scope of genome-editing tools. In this study, we found that SpCas9-NG not only targeted the genome but also efficiently self-targeted the single-guide RNA sequence in transfer DNA in transgenic plants, potentially increasing off-target risk by generating new single-guide RNAs. We further showed that the self-target effect of SpCas9-NG could be greatly alleviated by using a modified single-guide RNA scaffold starting with a GCCCC sequence.
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http://dx.doi.org/10.1038/s41477-020-0603-9DOI Listing
March 2020

A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice.

Plant Biotechnol J 2020 09 1;18(9):1845-1847. Epub 2020 Apr 1.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China.

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http://dx.doi.org/10.1111/pbi.13348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415777PMC
September 2020

Effect of multimedia-based nursing visit on perioperative anxiety in esophageal squamous cell carcinoma patients undergoing video-assisted thoracoscopic surgery.

Psychol Health Med 2019 12 25;24(10):1198-1206. Epub 2019 Mar 25.

Department of Nursing, The Affiliated Tumor Hospital of Nantong University , Nantong , Jiangsu Province , China.

Little is known about the multimedia-based preoperative nursing visit for squamous cell carcinoma (ESCC) patients undergoing video-assisted thoracoscopic surgery (VAST). The aim of this study was to evaluate the effects of preoperative multimedia-based nursing visit on perioperative anxiety in ESCC patients undergoing VAST. A total of 128 ESCC patients undergoing VAST were randomly divided into intervention group ( = 63) or control group ( = 65). The anxiety level was measured by state-trait anxiety inventory (STAI) and visual analog scale (VAS). The vital signs were also recorded. The data were collected at three different time points: before the intervention, 1 h before surgery and 24 h after surgery. There was no statistically significant difference in baseline STAI score, VAS scores and vital signs ( > 0.05). The intervention group reported significantly lower anxiety and improved vital signs in terms of systolic blood pressure, diastolic blood pressure and heart rate at 1 h before surgery and 24 h after surgery ( < 0.05). However, no significant difference in respiratory rate was observed between two groups at 1 h before surgery and 24 h after surgery ( > 0.05). Preoperative nursing visit with multimedia could reduce perioperative anxiety levels as well as help to stabilize vital sign for ESCC patients undergoing VAST.
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http://dx.doi.org/10.1080/13548506.2019.1595687DOI Listing
December 2019

Enhanced genome editing in rice using single transcript unit CRISPR-LbCpf1 systems.

Plant Biotechnol J 2019 03 19;17(3):553-555. Epub 2018 Nov 19.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China.

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http://dx.doi.org/10.1111/pbi.13028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381782PMC
March 2019

Isolation of five rice nonendosperm tissue-expressed promoters and evaluation of their activities in transgenic rice.

Plant Biotechnol J 2018 06 28;16(6):1138-1147. Epub 2017 Nov 28.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China.

Using promoters expressed in nonendosperm tissues to activate target genes in specific plant tissues or organs with very limited expression in the endosperm is an attractive approach in crop transgenic engineering. In this article, five putative nonendosperm tissue-expressed promoters were cloned from the rice genome and designated P , P , P , P and P . By qualitatively and quantitatively examining GUSplus reporter gene expression in transgenic rice plants, P -P were all found to be active in the roots, leaves, stems, sheaths and panicles but not in the endosperm of plants at different developmental stages. In addition, P , P and P were also inactive in rice embryos. Among these promoters, P and P exhibited higher activities in all of the tested tissues, and their activities in stems, leaves, roots and sheaths were higher than or comparable to those of the rice Actin1 promoter. We also progressively monitored the activities of P -P in two generations of single-copy lines and found that these promoters were stably expressed between generations. Transgenic rice was produced using P and P to drive a modified Bt gene, mCry1Ab. Bt protein expressed in the tested plants ranged from 1769.4 to 4428.8 ng/g fresh leaves, whereas Bt protein was barely detected in the endosperm. Overall, our study identified five novel nonendosperm tissue-expressed promoters that might be suitable for rice genetic engineering and might reduce potential social concern regarding the safety of GMO crops.
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http://dx.doi.org/10.1111/pbi.12858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978396PMC
June 2018

Isolation and identification of five cold-inducible promoters from Oryza sativa.

Planta 2018 Jan 6;247(1):99-111. Epub 2017 Sep 6.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, China.

Main Conclusion: Five promoters of the cold-inducible rice genes were isolated. The quantitative and qualitative expression analyses in the high generation transgenic rice suggest that the genes are stably induced by low temperature. Cold-inducible promoters are highly desirable for stress-inducible gene expression in crop genetic engineering. In this study, five rice genes, including OsABA8ox1, OsMYB1R35, OsERF104, OsCYP19-4, and OsABCB5, were found to be transcriptionally induced by cold stress. The promoters of these five genes were isolated, and their activities were identified in various tissues of transgenic rice plants at different growth stages both before and after cold stress. Histochemical staining, quantitative fluorescence assays, and GUSplus gene expression assays in corresponding promoter-GUSplus transgenic rice plants confirmed that the five promoters were cold-inducible with different expression patterns and strengths. The OsABA8ox1 and OsERF104 promoters had very low background expression; in contrast, the OsMYB1R35 promoter had higher basal activity in the roots, and OsCYP19-4 promoter activity was preferentially high in leaves and flowers of untreated transgenic lines. The OsABCB5 promoter had the highest basal activity among the five promoters. After cold induction, the activities of the OsABA8ox1, OsMYB1R35, and OsABCB5 promoters were high in both roots and leaves, slightly lower than that of the constitutively expressed OsActin1 promoter but comparable to that of the AtRD29A promoter. During the cold treatment time course, the activities of OsABA8ox1 and OsABCB5 promoters were quickly up-regulated in the early period and peaked at 24 h, after which the induction level gradually decreased until 48 h. The activities of the OsMYB1R35 and OsCYP19-4 promoters increased under stress in a time-dependent manner, while OsERF104 promoter activity began to increase at 4 h and then decreased strongly. Furthermore, activities' analysis in T, T, and T homozygous progeny of single-copy plants revealed that five promoters maintained their activities at comparable levels with no evidence of silencing under cold stress. Overall, the five cold-inducible rice promoters described herein could potentially be used in crop biotechnology.
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http://dx.doi.org/10.1007/s00425-017-2765-xDOI Listing
January 2018

Generation of targeted mutant rice using a CRISPR-Cpf1 system.

Plant Biotechnol J 2017 Jun 19;15(6):713-717. Epub 2017 Feb 19.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China.

CRISPR-Cpf1 is a newly identified CRISPR-Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR-Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR-Cpf1. We found that pre-crRNAs with a full-length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA-Cpf1-induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR-Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.
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http://dx.doi.org/10.1111/pbi.12669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425385PMC
June 2017

Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

Methods Mol Biol 2017 ;1498:33-40

Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, 40# Nongke South Road, Hefei, 230031, China.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.
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http://dx.doi.org/10.1007/978-1-4939-6472-7_3DOI Listing
January 2018

Research review on the pharmacological effects of astragaloside IV.

Fundam Clin Pharmacol 2017 Feb 23;31(1):17-36. Epub 2016 Sep 23.

Engineering Research Center of Chinese Traditional Veterinary Medicine, Beijing, China.

Astragalus membranaceus Bunge has been used to treat numerous diseases for thousands of years. As the main active substance of Astragalus membranaceus Bunge, astragaloside IV (AS-IV) also demonstrates the potent protective effect on focal cerebral ischemia/reperfusion, cardiovascular disease, pulmonary disease, liver fibrosis, and diabetic nephropathy. Based on studies published during the past several decades, the current state of AS-IV research and the pharmacological effects are detailed, elucidated, and summarized. This review systematically summarizes the pharmacological effects, metabolism mechanism, and the toxicity of AS-IV. AS-IV has multiple pharmacologic effects, including anti-inflammatory, antifibrotic, antioxidative stress, anti-asthma, antidiabetes, immunoregulation, and cardioprotective effect via numerous signaling pathways. According to the existing studies and clinical practices, AS-IV possesses potential for broad application in many diseases.
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http://dx.doi.org/10.1111/fcp.12232DOI Listing
February 2017

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice.

J Genet Genomics 2016 08 29;43(8):529-32. Epub 2016 Jul 29.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China; School of Agronomy, Anhui Agricultural University, Hefei 230036, China. Electronic address:

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http://dx.doi.org/10.1016/j.jgg.2016.07.003DOI Listing
August 2016

Plant phosphomannose isomerase as a selectable marker for rice transformation.

Sci Rep 2016 05 13;6:25921. Epub 2016 May 13.

Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, China.

The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering.
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http://dx.doi.org/10.1038/srep25921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865823PMC
May 2016

Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.

Rice (N Y) 2014 2;7(1). Epub 2014 May 2.

Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China.

Background: The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation.

Findings: Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.

Conclusions: Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.
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http://dx.doi.org/10.1186/s12284-014-0005-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052633PMC
June 2014
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