Abstract
Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems.
Highlights
We report an efficient system using truncated-gRNAs in the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] system to produce site-directed modifications in plants with no off-target effects
To evaluate mutation and off-target rates when using tru-gRNAs in plant cells, we designed 17–18 bp tru-gRNAs targeted towards several genes functioning in stress response and plant growth in Arabidopsis: ABSCISIC ACID INSENSITIVE4 (ABI4)[36], GLABRA1 (GL1)[37], and OST228 (Fig. 1b)
The selected tru-gRNAs for each gene were cloned into the CRISPR/Cas[9] vector described above, and the constructs were named ABI4-CRISPR, GL1-CRISPR1, GL1-CRISPR2, OPEN STOMATA 2 (OST2)-CRISPR1, and OST2-CRISPR2 (Fig. 1b)
Summary
We report an efficient system using truncated-gRNAs (tru-gRNAs) in the CRISPR/Cas[9] system to produce site-directed modifications in plants with no off-target effects. We focused on mutation of an abiotic stress-responsive factor in order to evaluate modification of a functional gene in plants, the gene encoding OPEN STOMATA 2 (OST2) (AHA1)—a major plasma membrane H+-ATPase in stomata response in Arabidopsis[28]. We applied our improved CRISPR/Cas[9] system to introduce novel alleles of OST2 in Arabidopsis using co-expression of Cas[9] and GFP via the 2A-coupled system to monitor and select Cas9-expressing cells[34]. The novel alleles for OST2 exibited altered stress responses in Arabidopsis These results suggest further applications of CRISPR/Cas9-mediated genome editing to improve plant growth and stress responses
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