scholarly journals Highly Efficient and Heritable Targeted Mutagenesis in Wheat via the Agrobacterium tumefaciens-Mediated CRISPR/Cas9 System

2019 ◽  
Vol 20 (17) ◽  
pp. 4257 ◽  
Author(s):  
Shujuan Zhang ◽  
Rongzhi Zhang ◽  
Jie Gao ◽  
Tiantian Gu ◽  
Guoqi Song ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Targeted Mutagenesis ◽  
Mutation Rates ◽  
Next Generation ◽  
Induced Mutations ◽  
D Genome ◽  
Agrobacterium Mediated Transformation ◽  
Biallelic Mutations ◽  
New Mutations

The CRISPR/Cas9 system has been successfully used in hexaploid wheat. Although it has been reported that the induced mutations can be passed to the next generation, gene editing and transmission patterns in later generations still need to be studied. In this study, we demonstrated that the CRISPR/Cas9 system could achieve efficient mutagenesis in five wheat genes via Agrobacterium-mediated transformation of an sgRNA targeting the D genome, an sgRNA targeting both the A and B homologues and three tri-genome guides targeting the editing of all three homologues. High mutation rates and putative homozygous or biallelic mutations were observed in the T0 plants. The targeted mutations could be stably inherited by the next generation, and the editing efficiency of each mutant line increased significantly across generations. The editing types and inheritance of targeted mutagenesis were similar, which were not related to the targeted subgenome number. The presence of Cas9/sgRNA could cause new mutations in subsequent generations, while mutated lines without Cas9/sgRNA could retain the mutation type. Additionally, off-target mutations were not found in sequences that were highly homologous to the selected sgRNA sequences. Overall, the results suggested that CRISPR/Cas9-induced gene editing via Agrobacterium-mediated transformation plays important roles in wheat genome engineering.

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.

scholarly journals Targeted mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family suggests competition of multiplexed sgRNAs for Cas9 apoprotein

2020 ◽  
Author(s):  
Juan Angulo ◽  
Christopher P Astin ◽  
Olivia Bauer ◽  
Kelan J Blash ◽  
Natalee M Bowen ◽  
...  
Keyword(s):  
Gene Family ◽  
Genetic Interaction ◽  
Targeted Mutagenesis ◽  
Mutation Rates ◽  
Colored Glass ◽  
Induced Mutations ◽  
Guide Rna ◽  
Fluorescent Markers ◽  
Bona Fide ◽  
Dna Vectors

AbstractGenome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas9 machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal lab settings, such as in the classroom or citizen science projects. Spacer sequences targeting genes of interest can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic as well as transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family, we found that Cas9 expression from an EGG CELL1 (EC1) promoter resulted in tenfold lower mutation rates than expression from a UBIQUITIN10 (UBQ10) promoter. A collection of bona fide null mutations in all nine GRF genes could be established with little effort. Finally, we explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. Multiplexing caused strong interference effects: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species. Our results suggest potential detrimental genetic interaction between induced mutations as well as competition of CRISPR RNAs for a limiting amount of Cas9 apoprotein.

scholarly journals CRISPR-Cas9 Gene Editing in Lizards Through Microinjection of Unfertilized Oocytes

2019 ◽  
Author(s):  
Ashley M. Rasys ◽  
Sungdae Park ◽  
Rebecca E. Ball ◽  
Aaron J. Alcala ◽  
James D. Lauderdale ◽  
...  
Keyword(s):  
Reproductive Biology ◽  
Gene Function ◽  
Gene Editing ◽  
Targeted Mutagenesis ◽  
Efficient Production ◽  
Direct Manipulation ◽  
Induced Mutations ◽  
New Approach ◽  
Evolution And Development

AbstractCRISPR-Cas9 mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are currently no reptiles with effective methods for targeted mutagenesis. Here we present a new approach that enables the efficient production of CRISPR-Cas9 induced mutations in Anolis lizards, an important model for studies of reptile evolution and development.

scholarly journals New mutations found by Next-Generation Sequencing screening of Spanish patients with Nemaline Myopathy

PLoS ONE ◽  
2018 ◽  
Vol 13 (12) ◽  
pp. e0207296 ◽  
Author(s):  
Sarah Moreau-Le Lan ◽  
Elena Aller ◽  
Ines Calabria ◽  
Lola Gonzalez-Tarancon ◽  
Cristina Cardona-Gay ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nemaline Myopathy ◽  
Next Generation ◽  
Generation Sequencing ◽  
New Mutations

scholarly journals Mutation rate analysis via parent–progeny sequencing of the perennial peach. II. No evidence for recombination-associated mutation

2016 ◽  
Vol 283 (1841) ◽  
pp. 20161785 ◽  
Author(s):  
Long Wang ◽  
Yanchun Zhang ◽  
Chao Qin ◽  
Dacheng Tian ◽  
Sihai Yang ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Recombination Rate ◽  
Mutation Rates ◽  
Recombination Rates ◽  
Rate Analysis ◽  
A Genome ◽  
Break Points ◽  
New Mutations

Mutation rates and recombination rates vary between species and between regions within a genome. What are the determinants of these forms of variation? Prior evidence has suggested that the recombination might be mutagenic with an excess of new mutations in the vicinity of recombination break points. As it is conjectured that domesticated taxa have higher recombination rates than wild ones, we expect domesticated taxa to have raised mutation rates. Here, we use parent–offspring sequencing in domesticated and wild peach to ask (i) whether recombination is mutagenic, and (ii) whether domesticated peach has a higher recombination rate than wild peach. We find no evidence that domesticated peach has an increased recombination rate, nor an increased mutation rate near recombination events. If recombination is mutagenic in this taxa, the effect is too weak to be detected by our analysis. While an absence of recombination-associated mutation might explain an absence of a recombination–heterozygozity correlation in peach, we caution against such an interpretation.

Discovery of mutations in Chenopodium quinoa Willd through EMS mutagenesis and mutation screening using pre-selection phenotypic data and next-generation sequencing

2018 ◽  
Vol 156 (10) ◽  
pp. 1196-1204 ◽  
Author(s):  
Camilo Mestanza ◽  
Ricardo Riegel ◽  
Santiago C. Vásquez ◽  
Diana Veliz ◽  
Nicolás Cruz-Rosero ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mutation Screening ◽  
Chenopodium Quinoa ◽  
Acetolactate Synthase ◽  
Screening Strategy ◽  
Next Generation ◽  
Induced Mutations ◽  
Phenotypic Data ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

AbstractQuinoa (Chenopodium quinoaWilld) is a dicotyledonous annual species belonging to the family Amaranthaceae, which is nutritionally well balanced in terms of its oil, protein and carbohydrate content. Targeting-induced local lesions in genomes (the TILLING strategy) was employed to find mutations in acetolactate synthase (AHAS) genes in a mutant quinoa population. TheAHASgenes were targeted because they are common enzyme target sites for five herbicide groups. Ethyl methane sulfonate (EMS) was used to induce mutations in theAHASgenes; it was found that 2% EMS allowed a mutation frequency of one mutation every 203 kilobases to be established. In the mutant population created, a screening strategy using pre-selection phenotypic data and next-generation sequencing (NGS) allowed identification of a mutation that alters the amino acid composition of this species (nucleotide 1231 codon GTT→ATT, Val→Ile); however, this mutation did not result in herbicide resistance. The current work shows that TILLING combined with the high-throughput of NGS technologies and an overlapping pool design provides an efficient and economical method for detecting induced mutations in pools of individuals.

scholarly journals Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells

2018 ◽  
Vol 215 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Akiko Seki ◽  
Sascha Rutz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Target Gene ◽  
Gene Knockout ◽  
Great Promise ◽  
Next Generation ◽  
Primary Mouse

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

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-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Seyed Mohammad Miri ◽  
Elham Tafsiri ◽  
William Chi Shing Cho ◽  
Amir Ghaemi
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Gene Editing ◽  
Genome Engineering ◽  
Building Blocks ◽  
Cell Receptor ◽  
Adoptive Cell Transfer ◽  
Promising Strategy ◽  
Pre Treatment ◽  
Cas A

Abstract Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called “adoptive cell transfer”, or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.

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