scholarly journals Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

2018 ◽  
Author(s):  
Shigeo S. Sugano ◽  
Ryuichi Nishihama ◽  
Makoto Shirakawa ◽  
Junpei Takagi ◽  
Yoriko Matsuda ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Genetic Analysis ◽  
Genome Editing ◽  
High Efficiency ◽  
Codon Optimization ◽  
Essential Gene ◽  
Land Plants ◽  
Marchantia Polymorpha ◽  
Model Species ◽  
Systematic Assessment

AbstractMarchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

scholarly journals Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

PLoS ONE ◽  
2018 ◽  
Vol 13 (10) ◽  
pp. e0205117 ◽  
Author(s):  
Shigeo S. Sugano ◽  
Ryuichi Nishihama ◽  
Makoto Shirakawa ◽  
Junpei Takagi ◽  
Yoriko Matsuda ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Genome Editing ◽  
Marchantia Polymorpha

scholarly journals Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants

2019 ◽  
Vol 116 (40) ◽  
pp. 20232-20239 ◽  
Author(s):  
Helge Berland ◽  
Nick W. Albert ◽  
Anne Stavland ◽  
Monica Jordheim ◽  
Tony K. McGhie ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Extreme Environments ◽  
Land Plant ◽  
Land Plants ◽  
Marchantia Polymorpha ◽  
Red Cell ◽  
Evolutionary Transition ◽  
Model Species ◽  
Harmful Effects ◽  
Early Land

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name “auronidin,” with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

scholarly journals Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants

2018 ◽  
Vol 218 (2) ◽  
pp. 554-566 ◽  
Author(s):  
Nick W. Albert ◽  
Amali H. Thrimawithana ◽  
Tony K. McGhie ◽  
William A. Clayton ◽  
Simon C. Deroles ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Genetic Analysis ◽  
Land Plants ◽  
Marchantia Polymorpha

scholarly journals One-shot generation of duodecuple (12x) mutant Arabidopsis: Highly efficient routine editing in model species

2020 ◽  
Author(s):  
Karen Barthel ◽  
Patrick Martin ◽  
Jana Ordon ◽  
Jessica L. Erickson ◽  
Johannes Gantner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Rna Polymerase Iii ◽  
Gene Families ◽  
Higher Order ◽  
Limiting Factor ◽  
Model Species ◽  
Guide Rna ◽  
Counter Selection ◽  
Transcriptional Units

SummaryGenome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Applications and Potential of Genome-Editing Systems in Rice Improvement: Current and Future Perspectives

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1359
Author(s):  
Javaria Tabassum ◽  
Shakeel Ahmad ◽  
Babar Hussain ◽  
Amos Musyoki Mawia ◽  
Aqib Zeb ◽  
...  
Keyword(s):  
Genome Editing ◽  
Crop Production ◽  
Grain Quality ◽  
High Efficiency ◽  
Abiotic Stress Tolerance ◽  
Mutation Breeding ◽  
Rice Grain ◽  
Rice Varieties ◽  
Rice Improvement ◽  
Breeding Techniques

Food crop production and quality are two major attributes that ensure food security. Rice is one of the major sources of food that feeds half of the world’s population. Therefore, to feed about 10 billion people by 2050, there is a need to develop high-yielding grain quality of rice varieties, with greater pace. Although conventional and mutation breeding techniques have played a significant role in the development of desired varieties in the past, due to certain limitations, these techniques cannot fulfill the high demands for food in the present era. However, rice production and grain quality can be improved by employing new breeding techniques, such as genome editing tools (GETs), with high efficiency. These tools, including clustered, regularly interspaced short palindromic repeats (CRISPR) systems, have revolutionized rice breeding. The protocol of CRISPR/Cas9 systems technology, and its variants, are the most reliable and efficient, and have been established in rice crops. New GETs, such as CRISPR/Cas12, and base editors, have also been applied to rice to improve it. Recombinases and prime editing tools have the potential to make edits more precisely and efficiently. Briefly, in this review, we discuss advancements made in CRISPR systems, base and prime editors, and their applications, to improve rice grain yield, abiotic stress tolerance, grain quality, disease and herbicide resistance, in addition to the regulatory aspects and risks associated with genetically modified rice plants. We also focus on the limitations and future prospects of GETs to improve rice grain quality.

scholarly journals Optimization of C-to-G base editors with sequence context preference predictable by machine learning methods

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tanglong Yuan ◽  
Nana Yan ◽  
Tianyi Fei ◽  
Jitan Zheng ◽  
Juan Meng ◽  
...  
Keyword(s):  
Machine Learning ◽  
High Efficiency ◽  
Codon Optimization ◽  
Sequence Context ◽  
Learning Methods ◽  
Specific Sequence ◽  
Uracil Dna Glycosylase ◽  
Base Editing ◽  
Machine Learning Methods ◽  
Deep Learning Model

AbstractEfficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.

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