- An expanded toolkit for precision genome engineering
- Identification of transgene-free CRISPR edited plants of rice and tomato by monitoring DsRED fluorescence in dry seeds
- Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing
- xCas9 expands the scope of genome editing with reduced efficiency in rice
- Developing a highly efficient and wildly adaptive CRISPR-SaCas9 toolset for plant genome editing
- Zinc finger nuclease‐mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template
- CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding
- CRISPR‐PLANT v2: An online resource for highly specific guide RNA spacers based on improved off‐target analysis
- Enhanced genome editing in rice using single transcript unit CRISPR‐LbCpf1 systems
- Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A
- Expanding the base editing scope in rice by using Cas9 variants
- Efficient Mitochondrial Genome Editing by CRISPR/Cas9
- Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining
- Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis
- Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice
- A CRISPR-based approach for targeted DNA demethylation
- Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana
- Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis
- Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration
- Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering
- Visualization analysis of CRISPR/Cas9 gene editing technology studies
- De novo domestication of wild tomato using genome editing
- Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system
- In vivo assembly of DNA-fragments in the moss, Physcomitrella patens
- A versatile and robust Agrobacterium‐based gene stacking system generates high‐quality transgenic Arabidopsis plants
- Multiplexed Gene Editing and Protein Overexpression Using a Tobacco mosaic virus Viral Vector
- A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
- A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants
- Improved Base Editor for Efficiently Inducing Genetic Variations in Rice with CRISPR/Cas9-Guided Hyperactive hAID Mutant
- Precise A·T to G·C Base Editing in the Rice Genome
- Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing
Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana
Abstract
Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes.Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes o fArabidopsis thalianaat frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1
plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold.Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I–SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions.
Full article [doi: 10.1111/tpj.12554]
