Targeted genome modification of crop plants using a CRISPR-Cas system

Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system

“…The OsCas9 was amplified from the vector provided by Shan et al (2013), and constructed into pCAMBIA2300-StU6p::sgRNA after 35S promoter by SpeI and BamHI to generate the final vector pCAM-BIA2300-35S::Cas9-StU6p::sgRNA. The guided RNA obtained by annealing two primers with the AarI complemented sequence at their 5 0 end was ligated into the final vector by AarI (Supplementary Fig.…”

“…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).…”

“…S1), which drove the sgRNA. The optimized coding sequence of SpCas9 in rice (OsCas9) (Shan et al 2013) driven by the CaMV 35S promoter was cloned into the same binary plasmid with StU6P::sgRNA (Fig. 1a).…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…In plants, the gene coding for the Cas9 protein from Streptococcus pyogenes (SpCas9) was codon-optimized for a good expression either in monocotyledonous or in dicotyledonous species [42,43]. The SpCas9 nuclease is the only Cas to have been used so far to induce gene knock-in in higher plants with efficiencies varying from 0.14 to 36% depending on the species, the delivery strategy and the transformation protocol (see Table 1).…”

“…So far, reports about CRISPR-assisted knock-in experiments in plants describe circular donor templates ranging from 0.47 to 5.2 kb in total length, including homology sequences of 46 bp to 1.6 kb on each side of the sequence of interest [2,6,8,10,43,67,68] ( Table 1). As for free DNA fragments, they varied from 72 to 476 bp including homology arms, of even length or not, of 46 to 146 bp [6,11,67,69].…”