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

Bae, Eun-Kyung; Choi, Hyunmo; Choi, Jin-Tak; Lee, Hyoshin; Kim, Sang-Gyu; Ko, Jae-Heung; Choi, Young‐Im

doi:10.1007/s11248-021-00272-9

Cited by 11 publications

(5 citation statements)

References 45 publications

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“…To avoid confusion and check the efficiency of the codon-optimized Cas9, we have designed and used single gRNA and their delivery in explants through a highly optimized Agrobacterium-mediated chickpea transformation method. Chimeric plants showed some green, and mostly the albino phenotypes in the same plants; and this was reported in many earlier studies (Gao et al, 2017; Odipio et al, 2017; Charrier et al, 2019; Pan et al, 2016; Li et al, 2013; Hooghvorst et al, 2019; Bae et al, 2021). Using a single sgRNA in the place of multiple sgRNAs was found better for gene editing and minimized the risk of off-target editing (Yu et al, 2018).…”

Section: Discussionsupporting

confidence: 85%

“…Knockout of the PDS gene by CRISPR/Cas9 moderated chlorophyll, carotenoid, and gibberellin biosynthesis leading to albino and dwarf phenotype (Qin et al , 2007). This study has been reported in melon (Hooghvorst et al , 2019), Medicago, cucumber (Chandrasekaran et al , 2016), watermelon (Tian et al , 2016), and hybrid poplar (Bae et al , 2021) with a single guide RNA (gRNA). This scorable marker, like the PDS gene, can better validate the feasibility of a gene-editing method as its disruption causes photobleaching or the albino phenotype in the transformed plants.…”

Section: Introductionmentioning

confidence: 72%

“…This study targeted the chickpea PDS gene in transformed stable chickpea plants. The PDS gene is generally used to optimize plants’ CRISPR/Cas9 system (Bae et al, 2021; Hooghvorst et al, 2019; Meng et al, 2017; Fan et al, 2015; Shan et al, 2013). This gene encodes a crucial enzyme in the carotenoid pathway, involving approximately twenty metabolic pathways.…”

Section: Discussionmentioning

confidence: 99%

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Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing

Gupta

Vishwakarma

Malakar

et al. 2022

Preprint

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Chickpea is considered recalcitrant to in vitro tissue culture. The Clustered, Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop rich in nutrients and protein. However, the generation of stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation approaches. We modified a binary vector pPZP200 by introducing a codon-optimized Cas9 gene for chickpea and the promoters of Medicago truncatula U6 snRNA for expressing guide RNA targeted to the Phytoene Desaturase (PDS) gene. The dissected single cotyledons with half embryo of chickpea were used as explants for genetic transformation. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. For the first time, this study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea phytoene desaturase gene (CaPDS) in stable shoots using an improved chickpea transformation protocol. Keywords: Chickpea transformation, CRISPR/Cas9, PDS, Codon optimization, Inference of CRISPR Edits (ICE)

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Section: Discussionsupporting

confidence: 85%

Section: Introductionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

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Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing

Gupta

Vishwakarma

Malakar

et al. 2022

Preprint

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“…However, as expected, there is no significant change in PagCSE1 expression in CSE2-sg3 poplar and vice versa (Figure 2b,c). This result is consistent to our previous report of PDS-CRISPR poplar study [29], and can be explained by nonsense-mediated mRNA decay, a surveillance pathway present in all eukaryotes, which eliminates mRNA transcripts containing premature stop codons, reducing gene expression errors [30].…”

Section: Predicted Cse Protein and Cse Gene Expression In Transgenic Cse-crispr Hybrid Poplarssupporting

confidence: 93%

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

Jang

Bae

Kim

et al. 2021

IJMS

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Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

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“…glandulosa) can tolerate flooding, has extensive root system, and has salt and cold tolerance (Qiu et al, 2019). Additionally, hybrid poplar has been used in many researches due to their high rates of transformation and well-documented genomic resources (Bae et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Knockdown of PagSAP11 Confers Drought Resistance and Promotes Lateral Shoot Growth in Hybrid Poplar (Populus alba × Populus tremula var. glandulosa)

et al. 2022

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Plants have evolved defense mechanisms to overcome unfavorable climatic conditions. The growth and development of plants are regulated in response to environmental stress. In this study, we investigated the molecular and physiological characteristics of a novel gene PagSAP11 in hybrid poplar (Populus alba × Populus tremula var. glandulosa) under drought stress. PagSAP11, a stress-associated protein (SAP) family gene, encodes a putative protein containing an A20 and AN1 zinc-finger domain at its N- and C-termini, respectively. Knockdown of PagSAP11 transgenic poplars (SAP11-Ri) enhanced their tolerance to drought stress compared with wild type plants. Moreover, the RNAi lines showed increased branching of lateral shoots that led to a gain in fresh weight, even when grown in the living modified organism (LMO) field. In SAP11-Ri transgenic plants, the expression levels of genes involved in axillary bud outgrowth and cell proliferation such as DML10, CYP707A and RAX were increased while the DRM gene which involved in bud dormancy was down-regulated. Taken together, these results indicate that PagSAP11 represents a promising candidate gene for engineering trees with improved stress tolerance and growth during unfavorable conditions.

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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

Cited by 11 publications

References 45 publications

Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing

Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

Knockdown of PagSAP11 Confers Drought Resistance and Promotes Lateral Shoot Growth in Hybrid Poplar (Populus alba × Populus tremula var. glandulosa)

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