CRISPR/Cas9 for Mutagenesis in Rice
TL;DR: This work presents a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.
Abstract: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.
Citations
More filters
TL;DR: In this paper , the authors used CRISPR/Cas9 to develop bio-fortified wheat using three homeologs in wheat designated as TaIPK1.A,TaIPK 1.B and TaIPk 1.D and found that the expression abundance of the homeolog was stronger in early stages of grain filling.
28 citations
TL;DR: In this article, CRISPR/Cas9-mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGC clusters.
Abstract: Rice (Oryza sativa) is a staple food crop and serves as a model cereal plant. It contains two biosynthetic gene clusters (BGCs) for the production of labdane-related diterpenoids (LRDs), which serve important roles in combating biotic and abiotic stress. While plant BGCs have been subject to genetic analyses, these analyses have been largely confined to the investigation of single genes. CRISPR/Cas9-mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGCs. Deletion of the BGC from chromosome 2 (c2BGC), which is associated with phytocassane biosynthesis, but not that from chromosome 4 (c4BGC), which is associated with momilactone biosynthesis, led to a lesion mimic phenotype. This phenotype is dependent on two closely related genes encoding cytochrome P450 (CYP) mono-oxygenases, CYP76M7 and CYP76M8, from the c2BGC. However, rather than being redundant, CYP76M7 has been associated with the production of phytocassanes, whereas CYP76M8 is associated with momilactone biosynthesis. Intriguingly, the lesion mimic phenotype is not present in a line with both BGCs deleted. These results reveal directional cross-cluster phytotoxicity, presumably arising from the accumulation of LRD intermediates dependent on the c4BGC in the absence of CYP76M7 and CYP76M8, further highlighting their interdependent evolution and the selective pressures driving BGC assembly.
9 citations
TL;DR: This work successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS and shows that simultaneous site-directed mutagenesis of two homoeologous loci by one sGRNA is successful.
Abstract: Soybean has a palaeopolyploid genome with nearly 75% of the genes present in multiple copies. Although the CRISPR/Cas9 system has been employed in soybean to generate site-directed mutagenesis, a systematical assessment of mutation efficiency of the CRISPR/Cas9 system for the multiple-copy genes is still urgently needed. Here, we successfully optimize one sgRNA CRISPR/Cas9 system in soybean by testing the efficiency, pattern, specificity of the mutations at multiple loci of GmFAD2 and GmALS. The results showed that simultaneous site-directed mutagenesis of two homoeologous loci by one sgRNA, the mutation frequency in the T0 generation were 64.71% for GmPDS, 60.0% for GmFAD2 and 42.86% for GmALS, respectively. The chimeric and heterozygous mutations were dominant types. Moreover, association of phenotypes with mutation pattern at target loci of GmPDS11 and GmPDS18 could help us further demonstrate that the CRISPR/Cas9 system can efficiently generate target specific mutations at multiple loci using one sgRNA in soybean, albeit with a relatively low transformation efficiency.
6 citations
02 Apr 2021
5 citations
TL;DR: It is shown that SlMAPK6 appears to regulate the synthesis of strigolactone and GA to induce the growth and development of tomato axillary buds.
Abstract: National Natural Science Foundation of China (No. 32060679; No.31760594; No. 31960604), Research Project on Talent Introduction of Guizhou University (No. [2017]50) and National Natural Fund Cultivation Project of Guizhou University (No. [2017] 5788-28).
2 citations
Cites background from "CRISPR/Cas9 for Mutagenesis in Rice..."
...…soybean, maize, wheat, sorghum and grape (Feng et al., 2014; Ito et al., 2015; Wang et al., 2016; Reem et al., 2019; Char et al., 2019a; Nishitani et al., 2016; Zhou et al., 2014; Tian et al., 2017; Char et al., 2019b; Di et al., 2019; Lee et al., 2019; Liang et al., 2019; Ren et al., 2019)....
[...]
...…apple, watermelon, potato, soybean, maize, wheat, sorghum and grape (Feng et al., 2014; Ito et al., 2015; Wang et al., 2016; Reem et al., 2019; Char et al., 2019a; Nishitani et al., 2016; Zhou et al., 2014; Tian et al., 2017; Char et al., 2019b; Di et al., 2019; Lee et al., 2019; Liang et…...
[...]
References
More filters
TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
12,865 citations
TL;DR: A method is presented for the rapid isolation of high molecular weight plant DNA which is free of contaminants which interfere with complete digestion by restriction endonucleases, and which yields total cellular DNA.
Abstract: A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
10,481 citations
TL;DR: In this paper, the authors describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions, and highlight challenges and future directions.
4,361 citations
TL;DR: A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens, and sequence analysis revealed that the boundaries of the T-DNA in transgenic Rice plants were essentially identical to those intransgenic dicotyledons.
Abstract: Summary
A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture
3,475 citations
01 Jun 2014
TL;DR: The development and applications of Cas9 are described for a variety of research or translational applications while highlighting challenges as well as future directions.
Abstract: Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.
3,270 citations