Abstract
CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5′-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.
Highlights
The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which originated as a bacterial adaptive immune system, is a programmable nuclease composed of a Cas[9] protein and a guide RNA molecule[1]
The 5′-untranslated region (5′-UTR) of certain mRNAs is known to act as a translation enhancer, largely increasing the production of the protein encoded by the downstream ORF5
The 5′-UTR from the alcohol dehydrogenase (ADH) gene has been applied as a translational enhancer in various studies focused on plant gene expression[6]
Summary
The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which originated as a bacterial adaptive immune system, is a programmable nuclease composed of a Cas[9] protein and a guide RNA (gRNA) molecule[1]. In addition to the high-level expression of the CAS9 gene, efficient translation of the mRNA may allow the generation of a large amounts of Cas[9] protein, resulting in increased efficiency toward the targeted mutagenesis. Solanum tuberosum L., has a tetraploid genome and commonly shows a vegetative reproduction manner This finding implies that there are large difficulties in obtaining homozygous mutants in which all of the target genes have been altered. A GBSSI-deficient mutant shows amylose-free starch in potato tubers. This phenotype is detected by visualization using the iodide staining method, as the starch properties are largely altered[16]. We have generated mutant potato alleles using GBSSI-targeted TALENs21
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