Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system

Abstract

Technologies to achieve the specific and precise knockout of genes are critical for understanding gene functions and fundamental biological processes. Targeted genome editing as a new and efficient method to mutate genes has been rapidly used in many organisms. Compared with the earlier systems, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 nuclease (Cas9) is an easier and more efficient system, and it has been widely used in recent years (Gaj et al. 2013). The Cas9 endonuclease is dictated by a 20-base pair (bp) sequence at the 50 end of the single-guide RNA (sgRNA), which then acts as a guide to the specific site of the genome, where Cas9 is able to cleave double-stranded DNA, leading to deletion, insertion, or substitution at the target sites (Sander and Joung 2014). Since 2013, CRISPR/Cas9 has been successfully applied by transient expression and/or stable transgenic lines in several plant species, such as Arabidopsis, Nicotiana benthamiana, rice, wheat, maize, and tomato (Brooks et al. 2014; Jiang et al. 2013; Li et al. 2013; Miao et al. 2013; Nekrasov et al. 2013; Shan et al. 2013). Moreover, the mutations generated in the primary transgenic plants by the CRISPR/Cas9 system can be stably transmitted to the next generation (Brooks et al. 2014; Feng et al. 2014). Thus, the CRISPR/Cas9 system is becoming a powerful tool for genome editing in plants, whereas the reports of the usage and efficiency of the CRISPR/Cas9 system-mediated plant genome engineering are still limited. Potato is a very important crop for world food security. Environmental changes and extended cultivation regions are challenges for potato cultivars. Understanding the functions of genes will help us to improve agronomic traits using molecular engineering technologies. Since cultivated potatoes are tetraploid, the functional approach of genes by molecular genetics is very difficult. Similar to other plant species in which the CRISPR/Cas9 has been successfully used, gene transformation in potato is efficient, and the genome sequence of double-haploid DM and diploid RH is available, which made potato an ideal candidate for this genome editing system (The Potato Genome Sequencing Consortium 2011). Here, we used the CRISPR/Cas9 system to produce knockouts of genes in potato successfully, which will provide an excellent foundation for future gene function studies. In order to construct a CRISPR/Cas9 plasmid which would function efficiently in potato, we first cloned a native promoter for potato U6 RNAs (StU6P) from DM Communicated by N. Stewart.