Genome editing technologies have become valuable tools in many aspects of scientific research, facilitating, for example, the identification of genes involved in development, or the determination of protein functions. They work by creating breaks within the genome, which are then repaired by re-joining the ends of the DNA. Errors often occur during this process, potentially leading to loss-of-function mutations. Additionally, as both strands of the DNA are broken, it provides an opportunity to incorporate new DNA sequences into the genome.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) system is one such method of genome editing. It has become difficult to ignore, having received a large amount of attention from both the scientific community and the media. One story that received significant coverage was the approval of a laboratory, based in London, to use CRISPR genome editing on human embryos. Several articles have also demonstrated the huge number of potential applications for this technique in animals, for example, producing cattle that do not develop horns and therefore avoid the need to undergo painful procedures to remove them. The reason for such excitement over CRISPR is, in part, due its simplicity and effectiveness.
The type II CRISPR system requires only a crisprRNA (crRNA), a trans-activating crRNA (tracrRNA), and a Cas9 protein. The RNA sequences can be fused to create a single guideRNA (gRNA). This can be reprogrammed by changing a 20 bp region known as the spacer sequence, to target the Cas protein to almost any region within the genome, referred to as the protospacer. The Cas9 protein recognises a 3 bp recognition sequence within the genome, known as a protospacer adjacent motif (PAM). This allows it to bind and introduce a break within the DNA sequence, exactly 3 bp upstream of the PAM (Belhaj et al., 2013). Unlike with alternative techniques for introducing DNA breaks, such as TALENs, various genomic regions can be easily targeted by simply modifying the RNA sequences used to target the Cas protein.
A recent study by Gao et al. (2016) attempted to design a way to effectively identify heritable mutations created using CRISPR/Cas9 in the T2 generation of Arabidopsis. Currently, used techniques are time-consuming and laborious. By including a fluorescent protein within the CRISPR plasmid, under a CRISPR/Cas9 vector promoter, the authors were able to easily screen seeds from the mutated plant that were still expressing the Cas9 protein using simple microscopy techniques. This is important when trying to determine if a mutation is heritable, as a T2 plant still expressing the CRISPR construct is likely to have a mutation due to the continued activity of Cas9 rather than having inherited it from the parent plant.
One significant disadvantage of the CRISPR system is the high levels of off-target mutations that can be introduced as a result. This makes it important to accurately determine whether the Cas9 gene has been segregated out, as plants that continue to express the Cas9 protein are much more likely to develop these undesirable modifications elsewhere in the genome. Furthermore, continued expression could lead to somatic mutations being falsely identified as heritable, which greatly increases the time needed to screen for plants containing the CRISPR/Cas9-derived mutations in subsequent generations.
Several studies have reported successful use of CRISPR/Cas9 in plants (Bortesi and Fischer, 2015), yet little emphasis was placed on ensuring if the Cas9 protein had been segregated out. Future studies, particularly those that require heritable mutations, should place more focus on those plants containing the mutation without continued expression of the CRISPR/Cas9 construct. This will significantly reduce the screening time for plants containing the CRISPR/Cas9-derived mutations as well as reduce the likelihood of off-target mutations elsewhere in the genome. Currently, this work was done using the popular model organism Arabidopsis, but it will be interesting to see whether such techniques can be expanded for use in other plant systems.
Khaoula Belhaj, Angela Chaparro-Garcia, Sophien Kamoun, Vladimir Nekrasov, 2013, 'Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system', Plant Methods, vol. 9, no. 1, p. 39 http://dx.doi.org/10.1186/1746-4811-9-39
Luisa Bortesi, Rainer Fischer, 2015, 'The CRISPR/Cas9 system for plant genome editing and beyond', Biotechnology Advances, vol. 33, no. 1, pp. 41-52 http://dx.doi.org/10.1016/j.biotechadv.2014.12.006
Xiuhua Gao, Jilin Chen, Xinhau Dai, Da Zhang, Yunde Zhao, 2016, 'An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing', Plant Physiology, p. pp.00663.2016 http://dx.doi.org/10.1104/pp.16.00663
Ganeshan Sivanandhan, Natesan Selvaraj, Yong Pyo Lim, Andy Ganapathi, 2016, 'Targeted Genome Editing Using Site-Specific Nucleases, ZFNs, TALENs, and the CRISPR/Cas9 system Takashi Yamamoto (ed.).', Annals of Botany, p. mcw089 http://dx.doi.org/10.1093/aob/mcw089