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Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA

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Abstract

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

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Funding

This work was supported in part by the National Institute of Forest Science (Project No. FG0702-2018).

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Authors and Affiliations

  1. Forest Bioresources Department, National Institute of Forest Science, 39 Onjeong-ro, Gwonseon-gu, Suwon, 16631, Korea

    Eun-Kyung Bae, Ji Won Choi, Hyoshin Lee & Young-Im Choi

  2. Forest Biomaterials Research Center, National Institute of Forest Science, 672 Jinju-daero, Jinju, 52817, Korea

    Hyunmo Choi

  3. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daehak-ro 291, Yuseong-gu, Daejeon, 34141, Korea

    Sang-Gyu Kim

  4. Plant and Environmental New Resources, Kyung Hee University, 1732 Deongyeong-daero, Giheung-gu, Yongin, 17104, Korea

    Jae-Heung Ko

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  1. Eun-Kyung Bae
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11248_2021_272_MOESM1_ESM.pptx

Figure S1. Position of target region of partial PagPDS1 gene. (a) Schematic of three exons and two introns and (b) sequence alignment of PagPDS1 genomic DNA and cDNA. Target sequences and PAM sites are indicated with underlined and blue bold letters, respectively. Boxes and lines indicate the exons and introns, respectively. Numbers of nucleotides are the beginning and end of each exon. Dotted line means the last part omitted. The primers used for mutation analysis are indicated by black bars above the nucleotide sequences (PPTX 77 kb)

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11248_2021_272_MOESM3_ESM.pptx

Figure S2. Summary of full-length cDNA sequences of the PagPDS1 and PagPDS2 alleles from P. alba (Pa) and P. glandulosa (Pg). Multiple alignments of (a) PagPDS1-Pa and PagPDS1-Pg with homologs from P. trichocarpa (PtrPDS1, Potri.014G148700) and (b) PagPDS2-Pa and PagPDS2-Pg with homologs from P. trichocarpa (PtrPDS21, Potri.002G235200) are shown. Multiple sequences of PDS genes were aligned using Clustal Omega (https://ebi.ac.uk/Tools/msa/clustalo/). Target sequences and PAM sites are indicated with underlined and blue bold letters, respectively. Asterisks (*) indicate homology between PDS genes from the two poplar species. The primers used for gene cloning and qRT-PCR analysis are indicated by black bars above the nucleotide sequences. The second exons of PagPDS1 and PagPDS2 are indicated by boxes. Green letters indicate single-nucleotide polymorphisms between the Pa and Pg alleles of PagPD1 and the PagPDS2 allele (PPTX 105 kb)

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Bae, EK., Choi, H., Choi, J.W. et al. Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA. Transgenic Res 30, 837–849 (2021). https://doi.org/10.1007/s11248-021-00272-9

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