CRISPR/Cas-mediated genome editing for crop improvement: current applications and future prospects

장규필,정영희 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.1

Conventional breeding techniques for crop improvement are based on hybridization and selection. However, due to the long breeding cycles of crops and the potentially unpredictable effects of traditional breeding, these techniques are not sufficient to meet market demands for crops with a variety of traits or to address the emerging food crisis we could face in the near future. In the past decade, advanced technologies such as next-generation sequencing have been used to rapidly produce massive amounts of genome sequence information in many crop species. These techniques, together with targeted genome editing tools such as Zinc Finger Nuclease (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Sequences (CRISPR)/CRISPR-associated protein (Cas) have increased the possibilities for crop improvement via targeted genome editing. The use of these technologies in crop biology has opened up a new era of genome editing-mediated crop breeding. In this review, we summarize the current techniques used for site-directed genome editing in plants, focusing on the CRISPR/Cas system, and discuss their current and future applications for crop biology.

Volatile methyl jasmonate is a transmissible form of jasmonate and its biosynthesis is involved in systemic jasmonate response in wounding

장규필,최양도,심재성,정춘균,송종태,이한용,정필중,김주곤 한국식물생명공학회 2014 Plant biotechnology reports Vol.8 No.6

Volatile organic compounds (VOCs) easilydiffuse due to their high hydrophobicity. Because of thisphysical property, VOCs are able to act as crucial signallingmolecules mediating intercellular and interplantcommunication. Methyl jasmonate (MeJA) is a volatileester form of jasmonic acid (JA) that is involved in interplantcommunication in response to biotic and abioticstresses. Despite its function in interplant communication,the specific role of MeJA in the regulation of intercellularjasmonate responses have been poorly understood. In thisstudy, we demonstrated that MeJA is much more effectivethan JA in inducing jasmonate response, and the higherefficacy of MeJA relies on its volatile property. Tounderstand the function of MeJA in the regulation of thejasmonate response, we analysed function of JMT gene,Jasmonic acid Methyl Transferase using its knockoutmutant (jmt) and overexpressing plants (35S:JMT). Mutantplants that lack JMT expression exhibited reduced jasmonateresponse, while JMT-overexpressing plants exhibiteda higher jasmonate response to JA treatment compared towild-type plants. In this study, we also showed that JMT isspecifically expressed in the phloem, the main vascularsystem for the transport of phytohormones, and that JMTexpression affects systemic jasmonate response inwounding. These results suggest the volatile MeJA is atransmissible form of jasmonate and that its biosynthesis isinvolved in systemic jasmonate response in wounding.

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

장규필,이상율,장선현,김주곤,최양도 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.4

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.

Genetic chimerism of CRISPR/Cas9-mediated rice mutants

장규필,Sangyool Lee,엄태영,Sun Hyun Chang,이한용,정필중,김주곤,최양도 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.6

The CRISPR/Cas9 technology is useful for genome editing to generate targeted mutants. Based on this genome editing technology, it was attempted to generate the rice mutant which lacks JAZ9 activity to understand its function in stress response. The sequence of guide RNA for the recognition of target site was obtained from CRISPRPLANT website (http://www.genome.arizona.edu/crispr) to minimize off-target effect and was recombined into the CRISPR/Cas9 binary vector pRGEB31. Embryonic calli regenerated from the mature seeds (O. sativa L. cv. Nakdong) were co-cultivated with transformed Agrobacterium tumefaciens LBA4404, and 26 individual transgenic plants were obtained through the hygromycin selection process. Nucleotide sequence analysis showed that most of T0 plants carried both edited and unedited wt sequence of JAZ9, suggesting genetic chimerism of T0 plants. Even though 2 individual lines carried homozygous mutation on JAZ9, they were also chimeric due to biallelic mutation. The relative ratio between edited and unedited wt sequence was variable among individual lines. Expression level of Cas9 is correlated with the frequency of genome editing frequency. In some plants, the enrichment ratio changed along with developmental stage. The nucleotide sequence analysis revealed that Cas9-mediated A/T addition occurred at -3 nucleotide position from protospacer adjacent motif (PAM), whereas G addition at -5 nucleotide position from the PAM. Further analysis of T1 transgenic plants showed that the genome editing patterns were similar between T0 plants and their T1 sibling plants. These suggested that earlier selection of T0 plants with homozygous mutation is an efficient way to obtain homozygous mutants in T1 generation.

The soluble ABA receptor PYL8 regulates drought resistance by controlling ABA signaling in Arabidopsis

이한용,장규필,엄태영,김주곤,이종섭,최양도 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.5

The phytohormone abscisic acid (ABA) mediates plant responses to environmental stresses, including drought stress. Recent identification of a family of soluble ABA receptors has expanded our understanding of ABA signaling in plants. Here, we examined the function of PYL8, which encodes a soluble ABA receptor. Plants overexpressing PYL8 (PYL8ox) showed lower germination rates and more inhibition of root growth than wild-type plants in response to ABA; PYL8ox plants also showed enhanced drought tolerance. By contrast, PYL8-silenced (PYL8i) plants showed less inhibition of root growth in response to ABA; PYL8i plants also showed decreased drought tolerance, compared with wild-type plants. The PYL8ox plants showed higher transcript levels of ABAresponsive genes and PYL8i plants showed lower transcript levels of these genes. Altered expression of PYL8 did not affect expression of other PYL family members. We also found that subcellular localization of PYL8 changed in response to ABA. These results suggest that PYL8 plays an essential role in ABA-mediated developmental processes and drought responses.

Segregation of genetic chimeras generated by CRISPR/Cas9 system in rice

이상열,김주곤,최양도,장규필 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.1

CRISPR/Cas9 system based on the adaptive immune system of bacteria is an emerging powerful technique of genome editing. Previously, we showed that transgenic rice transformed with a recombinant CRISPR/Cas9-OsJAZ9 binary plasmid display genetic chimerism in T0 generation, and the genetic chimerism includes homozygous and heterogeneous mutation of the target gene, OsJAZ9. The homozygous mutation of the T0 lines was stably transferred into the next generation, and all T1 plants of the T0 homozygous mutants were homozygous mutants. However, the fate of T1 plants with heterogeneous mutation remains poorly understood. In this study, we characterized target gene mutation in the progenies of the T0 lines with heterogeneous mutation to understand fate of the T0 lines with heterogeneous mutation. We found that some T0 lines such as lines 6 and 21 produce the T1 plants carrying homozygous mutation of OsJAZ9 through genetic segregation, while other T0 lines such as lines 3, 4 and 15 did not produce homozygous mutants in T1 generation unlike lines 6 and 21. These indicated that 40% of T0 lines with heterogeneous mutation produce the plants with homozygous mutation in T1 generation, suggesting that every T0 heterogeneous line does not produce the progenies with homozygous mutation through genetic segregation. However, because the portion of T0 lines with heterogeneous mutation (61%) is much bigger than that of T0 lines with homozygous mutation (8%) in T0 pool, these results propose that T0 heterogeneous mutants are useful to obtain CRISPR/Cas9-mediated homozygous mutants.

CHLORIDE CHANNEL 1 promotes drought tolerance in rice, leading to increased grain yield

엄태영,이상율,김주곤,장규필,최양도 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.4

Plant chloride channels (CLCs) localize to the plasma and organellar membranes; these channels play pivotal roles in the modulation of ion homeostasis and cell turgor. Recent studies have shown that the expression of CLCs is involved in plant responses to environmental stress. Here, we examined the rice (Oryza sativa) tonoplast-localized channel OsCLC1. OsCLC1 is preferentially expressed in roots, and therefore, we generated transgenic rice with root-specific overexpression of OsCLC1 (RCc3::OsCLC1). We also identified a T-DNA mutant line that lacks expression of OsCLC1 (osclc1). We found that RCc3::OsCLC1 rice plants showed increased tiller number and grain yield, whereas the osclc1 plants exhibited decreased tiller number and grain yield, compared with wild type. These observations suggest that expression of OsCLC1 affects rice growth and productivity. Furthermore, RCc3::OsCLC1 plants showed enhanced drought tolerance, leading to increased grain yield compared to wild-type plants grown under the same conditions. By contrast, osclc1 mutants exhibited reduced drought tolerance and productivity compared to wild-type plants. When expression of OsCLC1 was analyzed in drought, jasmonic acid (JA) or abscisic acid (ABA)-treated rice, expression of OsCLC1 was preferentially upregulated in roots in response to drought and JA, and was preferentially upregulated in shoots in response to ABA. Together with the finding that expression of OsCLC1 is positively correlated both with expressions of OsDREB1A and OsbHLH148, key transcription factors in drought and JA responses, respectively, these results suggest that OsCLC1 regulates drought tolerance in rice and JA signaling is involved in this process.

The promoter of tomato HISTIDINE DECARBOXYLASE A is fruit-specific, and its expression is stably maintained in fruits during ripening

김아영,김현민,마상훈,박서영,MAI THANH DAT,장규필,정영희 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.1

Identifying novel promoters with specific temporal and spatial expression patterns is crucial for crop biotechnology. In this study, we isolated a fruit-specific promoter in tomato, HISTIDINE DECARBOXYLASE A (SlHDC-A) promoter. Through RNA-seq and RT-PCR analysis, we found that SlHDC-A was predominantly expressed in fruits and that its expression was stable in fruits during ripening. These results suggest that the promoter of SlHDC-A might have the ability to determine fruit-specific gene expression. To test this possibility, we generated transgenic tomato transformed with SlHDC-A::GUS and 35S::GUS. Unlike 35S::GUS transgenic tomato with constitutive expression in various tissues, SlHDC-A::GUS transgenic plants showed fruit-specific expression of GUS. The intensity of GUS activity in fruits of SlHDC-A::GUS transgenic plants was approximately tenfold higher than that in fruits of 35S::GUS transgenic plants. The core region responsible for its fruit-specific expression was identified by promoter deletion analyses. Removal of the − 880 to − 577 region abolished the fruit-specific expression of SlHDC-A promoter. This suggests that the − 880 to − 577 region is the core region responsible for the fruit-specific expression of SlHDC-A. This finding was further supported by analysis of chimeric fusion promoter. Unlike 35S minimal promoter which had no activity to express GUS, the chimeric fusion promoter of the core region and 35S minimal promoter showed fruit-specific expression similar to intact SlHDC-A promoter. Collectively, these findings indicate that the promoter of SlHDC-A is fruit-specific and the − 880 to − 577 region is the core region of SlHDC-A promoter.