Abstract
Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.
Highlights
CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) technology surpasses other genome editing tools such as zinc finger nucleases (ZFNs), meganucleases and TAL effector nucleases (TALENs) due to numerous advantages in terms of cost, ease of use and efficiency of targeting DNA sequences [1].The CRISPR-Cas9 system is based on RNA-protein interactions involving a single noncoding guide RNA and the Cas9 nuclease derived from Streptococcus pyogenes, aureus or thermophilus
The tomato WVA106 genotype was transformed via Agrobacterium tumefaciens carrying the pDe-Cas9-NptII-dihydroflavonol 4reductase (DFR)#1-DFR#2 binary vector
The DFR gene was successfully deleted in 25% of the plantlets carrying the CAS9 gene
Summary
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) technology surpasses other genome editing tools such as zinc finger nucleases (ZFNs), meganucleases and TAL effector nucleases (TALENs) due to numerous advantages in terms of cost, ease of use and efficiency of targeting DNA sequences [1]. The CRISPR-Cas system is based on RNA-protein interactions involving a single noncoding guide RNA (sgRNA) and the Cas nuclease derived from Streptococcus pyogenes, aureus or thermophilus. Double-stranded breaks (DSBs) induced by the CRISPR-Cas system are repaired using either of two mechanisms: i) non-homologous end joining (either the classical non-homologous end-joining reaction C-NHEJ or an alternative end-joining reaction altEJ, called microhomology-mediated end joining, MMEJ), that can be error-prone and introduce mutations including deletions or ii) homology-directed repair (HDR) that needs a repair template with homology to the targeted sequence and can lead to precise gene knock-in.
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