Page 9 - S. Plant biology

Genome editing effect of the CRISPR/Cas9 system in hybrid poplar using
agrobacterium mediated transformation

Su Jin Park, Young-Im Choi*, Eun-Kyung Bae, Hyung-Ho Kim
Forest Bioresources Department, National Institute of Forest Science, Suwon, 16631, South Korea

BACKGROUND AIM

Engineered nucleases can effectively digest and mutate targeted genes in the In this study, we provide experimental evidences that the CRISPR/Cas9
plant genome. The CRISPR/CRISPR-associated protein 9 (Cas9) system can mediated genome editing system effectively induces targeted mutations of
induce gene editing at a high rate within months and can be effectively used for PDS gene in a hybrid poplar (P. alba × P. glandulosa), which comes from
long-lived trees. the crossing a female Populus alba and male P. glandulosa.
METHODS

Plant materials
The six-week-old hybrid poplar (P. alba × P. glandulosa) were used for transformation by Agrobacterium to introduce the CRISPR/Cas9 system.
gRNA site selection in PagPDS1
The gRNA to edit PagPDS1 was selected by Cas-Designer from CRISPR RGEN Tools (www.rgenome.net/cas-designer) (Park et al. 2015). The genome database of P. trichocarpa
(v3.1) was used as the reference sequence. The protospacer adjacent motif (PAM) sequence was set to 5'-NGG-3' of Streptococcus pyogenes Cas9 (SpCas9).
CRISPR/Cas9 vector construction
The binary vector pHSE401 (Addgene, USA) was used to express Cas9 and PagPDS1 gRNA in the hybrid poplar according to the method described by Xing et al. (2014).
Agrobacterium-mediated transformation of the hybrid poplar
Agrobacterium-mediated transformation was performed using the method described by Choi et al. (2005).
PagPDS1 mutant detection in the transgenic hybrid poplar
PagPDS1 gene mutation analysis in transgenic plants was performed with Sanger sequencing (Macrogen, Korea). Genomic DNA was isolated from the leaves of wild-type and
PagPDS1 mutants as templates for PCR amplification. The primers PagPDS1-F2 (5'-CTCTCAAGTTGCCTTGAGATG-3') and PagPDS1-R2 (5'-GCACAATCTCTCAAGAGTACATGC-
3') were used to amplify the PagPDS1 target sequence.

RESULTS

Fig. 2 Phenotypes of CRISPR/Cas9-induced mutant hybrid
poplars. (A) Multiple shoot regeneration from hybrid poplar
after Agrobacterium mediated transformation. (B) albino, (C)
Pale-green, (D) Green, without visible difference compared to
non-transgenic wild-type.
Table 1 Summary of CRISPR/Cas9-induced mutation types in hybrid Poplar
Phenotype Albino Pale green Green Total
No. of Plants 66 16 28 110
Plants (%) 60 14.5 25.5 100
No. of sequencing 29 7 4 40
Fig. 1 Schematic diagram of the CRISPR/Cas9 target sites in the poplar PDS gene and vector construction. (a) Multiple alignment of the coding sequence of PagPDS1 with PDS sequences from P.
tomentosa (PtoPDS) and P. trichocarpa (PtrPDS) showing the position of the guide RNA (gRNA) target sequence in the poplar PDS gene. The box indicates the second exon of PDS1. The gRNA-binding
site is underlined. PAM sequences are marked by blue bold letters. (b) Schematic map of the CRISPR/Cas9 vector used for stable Agrobacterium-mediated transformation in hybrid poplar.
Table 2 Comparison of target sequences of mutant lines induced by CRISPR/Cas9
Muta No. of
Phenotype Type Sequences Amino acid
tions plants
PagPDS1-Pa TGTATGGACTATCCAAGACCGGACCTTGATAACACGGTGAATTTCTTAGAGGCTGCCT PagPDS1-Pa CMDYPRPDLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA -
Wild type PagPDS1-Pg CVDYPRPDLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA
PagPDS1-Pg TGTGTGGACTATCCAAGACCGGACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT -
PagPDS1-Pa TGTATGGACTATCCAAGA.CCGGA---------------------------GGCTGCCT PagPDS1-Pa CMDYPRPEA----------ALLSSSFRSSPRPAKPLNVVIAGA -27
1 PagPDS1-Pg CVDYPRTGP* 5
PagPDS1-Pg TGTGTGGACTATCCAAGAACCGGACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT +1
PagPDS1-Pa TGTATGGACTATCCAAGA.------CCTTGATAACACGGTGAATTTCTTAGAGGCTGCCT PagPDS1-Pa CMDYPRP* -5
2 PagPDS1-Pg CVDYPRPGP* 4
Albino PagPDS1-Pg TGTGTGGACTATCCAAGACCCGGACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT PagPDS1-Pa CMDYPRRTLITR* +1
PagPDS1-Pa TGTATGGACTATCCAAGA-CGGACCTTGATAACACGGTGAATTTCTTAGAGGCTGCCT
-1
3 PagPDS1-Pg CVDYPR-DLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA 1
PagPDS1-Pg TGTGTGGACTATCCAAGA---GACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT -3
PagPDS1-Pa TGTATGGACTATCCAAGA.-CGGACCTTGATAACACGGTGAATTTCTTAGAGGCTGCCT PagPDS1-Pa CMDYPRRTLITR* -1
4 PagPDS1-Pg CVDYPR-DLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA 2
PagPDS1-Pg TGTGTGGACTATCCAAGATCCGGACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT +1
PagPDS1-Pa TGTATGGACTATCCAAGCCCGGACCTTGATAACACGGTGAATTTCTTAGAGGCTGCCT PagPDS1-Pa CMDYPSPDLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA 1 alt
Pale green PagPDS1-Pg CVDYPRPDLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA 3
PagPDS1-Pg TGTGTGGACTATCCAAGACCGGACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT -
PagPDS1-Pa TGTATGGACTATCCAAGACCGGACCTTGATAACACGGTGAATTTCTTAGAGGCTGCCT PagPDS1-Pa CMDYPRPDLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA -
Green PagPDS1-Pg CVDYPRPDLDNTVNFLEAALLSSSFRSSPRPAKPLNVVIAGA 4
PagPDS1-Pg TGTGTGGACTATCCAAGACCGGACCTTGATAACACGGTGAATTTCTTAGAAGCTGCCT -
Target sequences with the PAM sequence highlighted in blue are underlined in the wild-type sequence. The mutations are indicated in red. Deletion and blank bases are indicated by red dashes and black dots, respectively. n.d. is not determined.
The mutations column indicates the number of inserted (+), deleted (-), and altered (alt) bases. PagPDS1.1a is one allele of the PDS1 gene from P. alba. PagPDS1.2a is another allele of the PDS1 gene from P. glandulosa.
Predicted proteins of PagPDS1 mutant lines. Target and PAM sequences are marked by black and blue bold letters, respectively. Replacement or deletion amino acids are indicated by red letters.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
The mutation rates of the albino, pale green, and green Choi Y-I, Noh EW, Lee H, Han MS, Lee JS, Choi KS (2005) This work was supported in part by the National Institute
phenotypes were 60%, 14.55%, and 25.45%, An efficient and novel plant selectable marker based on of Forest Science (Project No. FG0702-2018).
respectively. The albino phenotypes were found when organomercurial resistance. J Plant Biotechnol 48:351-355.
the mutation occurred in bi-alleles of the hybrid poplar. Park J, Bae S, Kim J-S (2015) Cas-Designer: A web-based Contact information
Collectively, the results show that the CRISPR/Cas9 tool for choice of CRIPSR-Cas9 target sites. Bioinformatics
system was effective in reducing gene expression and 31:4014-4016. *Corresponding authors: Young-Im Choi
altering the plant phenotype by editing a hybrid poplar E-mail: yichoi99@korea.kr
target gene with two alleles. We expect that this Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Address: Forest Bioresources Department, National Institute of
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technique will be used to improve tree quality. multiplex genome editing in plants. BMC Plant Biol 14:327. Forest Science, 39 Onjeong-ro, Gwonseon-gu, Suwon, 16631,
Korea