Abstract
Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] system is highly efficient at generating targeted mutations in stable transgenic poplar plants, and homozygous mutations at the desired sites can be created in the first generation
Since the whole-genome sequence of Populus trichocarpa was released in 200616, extensive genomic resources are available for functional genomics studies in this species, which has been used as a model in forest genetics and woody plant studies
In order to test whether the CRSPR/Cas[9] system could effectively direct gene-specific editing in Populus, we selected the poplar phytoene desaturase gene (PtoPDS), which is required for chlorophyll biosynthesis and its mutant shows an albino phenotype in other plant species[9,24], as the target of Cas[9] endonuclease
Summary
The CRISPR/Cas[9] system is highly efficient at generating targeted mutations in stable transgenic poplar plants, and homozygous mutations at the desired sites can be created in the first generation. There are three main types of engineered nucleases for genome editing: zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas[1]. The stable transformation process is applied to create genetically 1 edited lines carrying a mutation in the gene of interest in all these plant species using CRISPR/Cas[94]. We report an improved approach to delivery Cas[9] coding gene and multiple sgRNAs into plant cells by a single plasmid. Based on this system, an poplar endogenous phytoene desaturase gene (PtoPDS) was disrupted site- in the first generation of transgenic plants. Our data suggest that the CRISPR/Cas[9] system is a highly efficient and powerful tool for genome modifications in woody plants
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