scholarly journals Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System

2018 ◽  
Vol 19 (10) ◽  
pp. 3000 ◽  
Author(s):  
Shouhong Zhu ◽  
Xiuli Yu ◽  
Yanjun Li ◽  
Yuqiang Sun ◽  
Qianhao Zhu ◽  
...  
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Targeted Mutagenesis ◽  
Cotton Fibers ◽  
Efficient Tool ◽  
Gene Encoding ◽  
Editing Event ◽  
Highly Efficient ◽  
Target Sites ◽  
Targeted Gene Editing

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4–100% and 92.9–100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.

scholarly journals CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Changtian Pan ◽  
Lei Ye ◽  
Li Qin ◽  
Xue Liu ◽  
Yanjun He ◽  
...  
Keyword(s):  
Average Frequency ◽  
Transformation Method ◽  
Targeted Mutagenesis ◽  
Efficient Tool ◽  
Tomato Plants ◽  
High Frequencies ◽  
High Mutation Frequency ◽  
Transgenic Tomato Plants ◽  
Targeted Gene Editing ◽  
The Stability

Abstract The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants.

scholarly journals Generation and molecular characterization of CRISPR/Cas9-induced mutations in 63 immunity-associated genes in tomato reveals specificity and a range of gene modifications

2019 ◽  
Author(s):  
Ning Zhang ◽  
Holly M. Roberts ◽  
Joyce Van Eck ◽  
Gregory B. Martin
Keyword(s):  
Molecular Characterization ◽  
Gene Editing ◽  
Induced Mutations ◽  
Tomato Plants ◽  
Transgenic Tomato Plants ◽  
Target Sites ◽  
Targeted Gene Editing ◽  
Gene Alterations ◽  
New Mutations

AbstractThe CRISPR/Cas9 system is a powerful tool for targeted gene editing in many organisms including plants. However, most of the reported uses of CRISPR/Cas9 in plants have focused on modifying one or a few genes, and thus the overall specificity, types of mutations, and heritability of gene alterations remain unclear. Here we describe the molecular characterization of 361 T0 transgenic tomato plants that were generated using CRISPR/Cas9 to induce mutations in 63 immunity-associated genes. Among the T0 transformed plants, 245 carried mutations (68%), with 20% of those plants being homozygous for the mutation, 30% being heterozygous, 32% having two different mutations (biallelic) and 18% having multiple mutations (chimeric). The mutations were predominantly short insertions or deletions, with 87% of the affected sequences being smaller than 10 bp. The majority of 1 bp insertions were A (50%) or T (29%). The mutations from the T0 generation were stably transmitted to later generations, although new mutations were detected in some T1 plants. No mutations were detected in 18 potential off-target sites among 144 plants. Our study provides a broad and detailed view into the effectiveness of CRISPR/Cas9 for genome editing in an economically important plant species.

Prediction of off-target effects in crispr/cas9 system by ensemble learning

2021 ◽  
Vol 16 ◽  
Author(s):  
Yongxian Fan ◽  
Haibo Xu
Keyword(s):  
Gene Editing ◽  
Low Cost ◽  
Target Prediction ◽  
Optimal Parameters ◽  
Practical Application ◽  
Gene Encoding ◽  
Targeted Gene Editing ◽  
Target Effect ◽  
New Generation ◽  
Target Effects

Background: CRISPR/Cas9, a new generation of targeted gene editing technology with low cost and simple operation has been widely employed in the field of gene editing. The erroneous cutting of off-target sites in CRISPR/Cas9 is called off-target effect, which is also the biggest complication that CRISPR/Cas9 confronts in practical application. To be specific, the off-target effects could lead to unexpected gene editing results. Therefore, accurately predicting CRISPR/Cas9 off-target effect is a very important task. Predicting off-target effects of CRISPR/Cas9 by machine learning method is feasible, but most existing off-target tools did not pay close attention to the effects of gene encoding on prediction. Methods: We compared three encoding methods based on One-Hot and combined the gene sequence with four CRISPR/Cas9 off-target prediction tools to build an ensemble model with XGBoost, designated as XGBCRISPR. The grid search is employed to find the optimal parameters to achieve the best performance. Results: The performance is compared with existing tools based on the ROC value and PRC value. The experimental results show that the XGBCRISPR model is superior to the existing tools. Conclusion: The new model could achieve better prediction result than existing tools, but the accuracy of model can be improved further as many off-target scores appear.

scholarly journals Highly efficient multiplex genome editing in dicot plants using improved CRISPR/Cas systems

2022 ◽  
Author(s):  
Bingjie Li ◽  
Yun Shang ◽  
Lixianqiu Wang ◽  
Jing Lv ◽  
Fengjiao Wang ◽  
...  
Keyword(s):  
Life Span ◽  
Genome Editing ◽  
Gene Function ◽  
Gene Editing ◽  
Gene Promoter ◽  
Highly Efficient ◽  
Target Sites

CRISPR/Cas9-mediated gene editing provides a powerful tool for dissecting gene function and improving important traits in crops. However, there are still persisting challenges to obtain high homozygous/bi-allelic (ho/bi) mutations in dicot plants. Here, we develop an improved CRISPR/Cas9 system harboring a calreticulin-like gene promoter, which can boost targeted mutations in dicots. Additionally, the pDC45_dsg construct, combining a 35Spro-tRNA_sgRNA-EU unit and PCE8pro-controlled Cas9, can achieve more than 80.0% ho/bi mutations at target sites in allotetraploid tobacco. We construct pDC45_Fast system that can simultaneously fulfill gene editing and shorten the life span of T0 generation tobacco and tomato. This study provides new tools for improving targeted gene mutagenesis in dicots, and makes manipulations of genes in Solanum more feasible.

scholarly journals Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9

2021 ◽  
Vol 22 (13) ◽  
pp. 6989
Author(s):  
Yuya Liang ◽  
Sudip Biswas ◽  
Backki Kim ◽  
Julia Bailey-Serres ◽  
Endang M. Septiningsih
Keyword(s):  
Gene Editing ◽  
Indica Rice ◽  
Crop Improvement ◽  
Immature Embryo ◽  
Test Case ◽  
Submergence Tolerance ◽  
Embryogenic Calli ◽  
Submergence Stress ◽  
Efficient Vector ◽  
Vector Delivery

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

scholarly journals Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jonathan O’Keeffe Ahern ◽  
Irene Lara-Sáez ◽  
Dezhong Zhou ◽  
Rodolfo Murillas ◽  
Jose Bonafont ◽  
...  
Keyword(s):  
Delivery System ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Attractive Alternative ◽  
Safe Delivery ◽  
Guide Rna ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Targeted Gene Editing ◽  
Genomic Editing ◽  
Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

scholarly journals Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Akihiro Yasue ◽  
Silvia Naomi Mitsui ◽  
Takahito Watanabe ◽  
Tetsushi Sakuma ◽  
Seiichi Oyadomari ◽  
...  
Keyword(s):  
Mouse Embryos ◽  
Targeted Mutagenesis ◽  
Highly Efficient

Scalable Sulfonate-Coated Cotton Fibers as Facile Recyclable and Biodegradable Adsorbents for Highly Efficient Removal of Cationic Dyes

2021 ◽  
Author(s):  
Linhua Li ◽  
Baojie Dou ◽  
Jianwu Lan ◽  
Jiaojiao Shang ◽  
Yafang Wang ◽  
...  
Keyword(s):  
Adsorption Capacity ◽  
Large Scale ◽  
Ph Value ◽  
Cationic Dyes ◽  
Cotton Fibers ◽  
Wastewater Remediation ◽  
Efficient Removal ◽  
Adsorption Sites ◽  
Highly Efficient ◽  
Coated Cotton

Abstract Adsorbents with superior adsorption capacity and facile recyclability are viewed as promising materials for dye wastewater treatment. In this work, a novel sulfonate decorated cotton fiber as a biodegradable and recyclable adsorbent was fabricated for highly efficient removal of cationic dyes. Herein, the poly(sodium p-styrenesulfonate-co-N-methylol acrylamide) (P(SSNa-co-NMAM)) with SSNa units as adsorption sites and NMAM units as thermal-crosslinking points was synthesized for modification of cotton fibers in a large scale at high temperature (160 oC). The various characterization investigations confirmed the successful construction of the P(SSNa-co-NMAM) coated cotton fibers (PCF). As expected, the as-obtained adsorbent presented outstanding adsorption performance toward cationic dyes in the both static and dynamic states, even in the synthetic effluent. The adsorption processes of cationic dyes onto the PCF were well fitted by the Langmuir isotherm model and Pseudo-second-order kinetics, respectively. The thermodynamics study showed that the adsorption reaction of the cationic dyes onto PCF was a spontaneous and endothermic process. The maximum adsorption capacities of PCF toward MEB, RhB and MG were 3976.10, 2879.80 and 3071.55 mg/g, respectively. The responsible adsorption of dyes ontothe PCF was electrostatic interaction. Moreover, the adsorption capacity of PCF toward cationic dyes was slight influenced by pH value of solution, because of the stable feature of sulfonate moiety in acid and alkali. In addition, the as-prepared PCF exhibited satisfactory recyclability and reusability. Given the aforementioned results, the as-obtained PCF is a promising adsorbent with great potential for practical application in the dye-contaminated wastewater remediation.

CRISPR/Cas9-induced β-carotene Hydroxylase Mutation in Dunaliella Salina CCAP19/18

2021 ◽  
Author(s):  
Lina Hu ◽  
Shu ying FENG ◽  
Gaofeng Liang ◽  
Jingxia Du ◽  
Aifang Li ◽  
...  
Keyword(s):  
High Performance ◽  
Dunaliella Salina ◽  
Gene Editing ◽  
Expression System ◽  
Transformation Method ◽  
Practical Significance ◽  
Exon 1 ◽  
Targeted Gene Editing ◽  
Foreign Genes ◽  
Β Carotene

Abstract Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of CRISPR/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

Sign in / Sign up

Export Citation Format

Share Document