Figure 6
From: Targeted mutagenesis in wheat microspores using CRISPR/Cas9
CRISPR/Cas9-based editing of an endogenous ubiquitin gene in wheat microspores. (A) Schematic diagram of the pEEE005-NLSCas9/gUbi1 and pEEE006-NLSCas9/gUbi1/GFP constructs used for mutagenesis of in wheat microspores. (B) PCR/restriction enzyme assay to detect mutations in TaUbiL1 induced by Cas9/ gUbi1. (C) Deletions detected at the cleavage site in sequenced products of all three homoeologs of TaUbiL1 from wheat microspores transfected with pEEE005-NLSCas9/gUbi1 or pEEE006-NLSCas9/gUbi1/GFP constructs. The three homoeologues of TaUbiL1 were defined based on multiple single nucleotide polymorphisms in each background (sequence not shown) for the three genomes in this locus. (D) Sanger sequencing electropherograms showing mutations in TaUbiL1 homoeologues from the A, B and D genomes of wheat.
