Abstract
Targeted genome editing with the CRISPR/Cas9 system has been used extensively for the selective mutation of plant genes. Here we used CRISPR/Cas9 to disrupt the putative barley (Hordeum vulgare cv. “Golden Promise”) endo-N-acetyl-β-D-glucosaminidase (ENGase) gene. Five single guide RNAs (sgRNAs) were designed for different target sites in the upstream part of the ENGase coding region. Targeted fragment deletions were induced by co-bombarding selected combinations of sgRNA with wild-type cas9 using separate plasmids, or by co-infection with separate Agrobacterium tumefaciens cultures. Genotype screening was carried out in the primary transformants (T0) and their T1 progeny to confirm the presence of site-specific small insertions and deletions (indels) and genomic fragment deletions between pairs of targets. Cas9-induced mutations were observed in 78% of the plants, a higher efficiency than previously reported in barley. Notably, there were differences in performance among the five sgRNAs. The induced indels and fragment deletions were transmitted to the T1 generation, and transgene free (sgRNA:cas9 negative) genome-edited homozygous ENGase knock outs were identified among the T1 progeny. We have therefore demonstrated that mutant barley lines with a disrupted endogenous ENGase and defined fragment deletions can be produced efficiently using the CRISPR/Cas9 system even when this requires co-transformation with multiple plasmids by bombardment or Agrobacterium-mediated transformation. We confirm the specificity and heritability of the mutations and the ability to efficiently generate homozygous mutant T1 plants.
Highlights
Several platforms exist for specific genome editing using designer nucleases, including zinc finger nucleases (ZFNs; Bibikova et al, 2003), transcription activator-like effector nucleases (TALENs; Boch et al, 2009), and the clustered, regularly interspaced, short palindromic repeats (CRISPR) system, often paired with Cas9, the CRISPR-associated protein from Streptococcus pyogenes (Barrangou et al, 2007; Brouns et al, 2008; Garneau et al, 2010)
As a prerequisite step to produce knock out lines for the putative barley ENGase gene (MLOC_10039.2), its partial sequence of 616 bp was amplified from wild-type barley genomic DNA
Our data suggest that the CRISPR/Cas9 system generated double-stranded break (DSB) and subsequent mutations in the stably transformed barley plants with an efficiency of at least 78%
Summary
Several platforms exist for specific genome editing using designer nucleases, including zinc finger nucleases (ZFNs; Bibikova et al, 2003), transcription activator-like effector nucleases (TALENs; Boch et al, 2009), and the clustered, regularly interspaced, short palindromic repeats (CRISPR) system, often paired with Cas, the CRISPR-associated protein from Streptococcus pyogenes (Barrangou et al, 2007; Brouns et al, 2008; Garneau et al, 2010). Unlike ZFNs and TALENs, which are dimeric nucleases whose target specificity requires DNA–protein interactions, Cas is an RNAguided endonuclease whose specificity depends on the sequence of its single guide RNA (sgRNA). The latter hybridizes to a 20-nt (nucleotide) complementary DNA target (the protospacer), and Barley Fragment Deletions by CRISPR/Cas catalyzes a double-stranded break (DSB) 3–4 bp upstream of the protospacer adjacent motif (PAM), a short and degenerate sequence (5 -NGG-3 or 5 -NAG-3 for S. pyogenes Cas9) which is required for Cas to recognize the protospacer. Whereas different ZFNs and TALENs must be produced for different targets, the same Cas enzyme can be used to achieve any modification and only the sgRNA sequence must be changed. The sgRNA tolerates a certain number of mismatches so appropriate target sites need to be chosen to minimize unwanted off-target mutations (Pattanayak et al, 2013; Bae et al, 2014)
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