The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas–mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3′ end of the target site abolished the cleavage activity completely. The mismatches at the 5′ end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3′ end. This approach provides a powerful method for genome engineering in plants.

CRISPR/Cas9 genome editing technology is a DNA manipulation tool for trait improvement. This technology has been demonstrated and successfully applied to edit the genome in various species of plants. The delivery of CRISPR/Cas9 components within rigid plant cells is very crucial for high editing efficiency. Here, we insight the strengths and weaknesses of each method of delivery. The mutation efficiency of genome editing may vary and affected by different factors. Out of various factors, the delivery of CRISPR/Cas9 components into cells and genome is vital. The way of delivery defines whether the edited plant is transgenic or transgene-free. In many countries, the transgenic approach of improvement is a significant limitation in the regulatory approval of genetically modified crops. Gene editing provides an opportunity for generating transgene-free edited genome of the plant. Nevertheless, the mode of delivery of the CRISPR/Cas9 component is of crucial importance for genome modification in plants. Different delivery methods such as Agrobacterium-mediated, bombardment or biolistic method, floral-dip, and PEG-mediated protoplast are frequently applied to crops for efficient genome editing. We have reviewed different delivery methods with prons and cons for genome editing in plants. A novel nanoparticle and pollen magnetofection-mediated delivery systems which would be very useful in the near future. Further, the factors affecting editing efficiency, such as the promoter, transformation method, and selection pressure, are discussed in the present review.

Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34–22% reduction of the phytic acid content in the mature grains (T₄ seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals.