Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Eid, Ayman; Mohan, Chakravarthi; Sánchez, Sara S.; Wang, Duoduo; Altpeter, Fredy

doi:10.3389/fgeed.2021.654996

Cited by 44 publications

(41 citation statements)

References 48 publications

(65 reference statements)

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“…Precision genome editing of crops via HDR-mediated nucleotide substitutions has rarely been reported (Huang and Puchta, 2019 ). By contrast, loss-of-function mutations via the error-prone NHEJ repair pathway have been described in many crops (Yin et al, 2017 ; Huang and Puchta, 2019 ; Mao et al, 2019 ) including sugarcane (Jung and Altpeter, 2016 ; Kannan et al, 2018 ; Eid et al, 2021 ). Homology-directed repair frequency, as calculated as a percentage of GT events to all generated events, is typically in the range of 0.1 to a few percent (D'Halluin et al, 2013 ; Schiml et al, 2014 ; Svitashev et al, 2015 ; Endo et al, 2016 ; Hahn et al, 2018 ), which is more than an order of magnitude below the frequencies reported for NHEJ-mediated knock-outs of gene function (Yin et al, 2017 ; Hua et al, 2019 ; Huang and Puchta, 2019 ; Mao et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

et al. 2021

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Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

et al. 2021

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“…Recently, Eid et al (2021) reported efficient multi-allelic editing in sugarcane of the magnesium chelatase I subunit ( MgCh ), a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of MgCh was confirmed via Sanger sequencing and resulted in severely reduced chlorophyll content.…”

Section: Genome Editingmentioning

confidence: 99%

Genetic Transformation of Sugarcane, Current Status and Future Prospects

et al. 2021

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Sugarcane (Saccharum spp.) is a tropical and sub-tropical, vegetative-propagated crop that contributes to approximately 80% of the sugar and 40% of the world’s biofuel production. Modern sugarcane cultivars are highly polyploid and aneuploid hybrids with extremely large genomes (>10 Gigabases), that have originated from artificial crosses between the two species, Saccharum officinarum and S. spontaneum. The genetic complexity and low fertility of sugarcane under natural growing conditions make traditional breeding improvement extremely laborious, costly and time-consuming. This, together with its vegetative propagation, which allows for stable transfer and multiplication of transgenes, make sugarcane a good candidate for crop improvement through genetic engineering. Genetic transformation has the potential to improve economically important properties in sugarcane as well as diversify sugarcane beyond traditional applications, such as sucrose production. Traits such as herbicide, disease and insect resistance, improved tolerance to cold, salt and drought and accumulation of sugar and biomass have been some of the areas of interest as far as the application of transgenic sugarcane is concerned. Although there have been much interest in developing transgenic sugarcane there are only three officially approved varieties for commercialization, all of them expressing insect-resistance and recently released in Brazil. Since the early 1990’s, different genetic transformation systems have been successfully developed in sugarcane, including electroporation, Agrobacterium tumefaciens and biobalistics. However, genetic transformation of sugarcane is a very laborious process, which relies heavily on intensive and sophisticated tissue culture and plant generation procedures that must be optimized for each new genotype to be transformed. Therefore, it remains a great technical challenge to develop an efficient transformation protocol for any sugarcane variety that has not been previously transformed. Additionally, once a transgenic event is obtained, molecular studies required for a commercial release by regulatory authorities, which include transgene insertion site, number of transgenes and gene expression levels, are all hindered by the genomic complexity and the lack of a complete sequenced reference genome for this crop. The objective of this review is to summarize current techniques and state of the art in sugarcane transformation and provide information on existing and future sugarcane improvement by genetic engineering.

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“…Nevertheless, the CRISPR-Cas9 system offers an opportunity to generate the efficient multiallelic co-editing of a large number of copies of genes. Higher efficiency of editing can be achieved through optimization of the genome-editing reagents and their delivery to enable the efficient co-editing of a large number of copies/alleles ( Eid et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

“…Cana Flex I plants facilitate ethanol production and better extraction of other bioproducts. Eid et al (2021) used the CRISPR system to target the multiallelic magnesium chelatase subunit I ( MgCh ) gene, which is a key gene in chlorophyll biosynthesis. The CRISPR-Cas9 system could target 49 copies/alleles of magnesium chelatase, as confirmed via Sanger sequencing.…”

Section: Introductionmentioning

confidence: 99%

Recent Advances in Sugarcane Genomics, Physiology, and Phenomics for Superior Agronomic Traits

et al. 2022

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Advances in sugarcane breeding have contributed significantly to improvements in agronomic traits and crop yield. However, the growing global demand for sugar and biofuel in the context of climate change requires further improvements in cane and sugar yields. Attempts to achieve the desired rates of genetic gain in sugarcane by conventional breeding means are difficult as many agronomic traits are genetically complex and polygenic, with each gene exerting small effects. Unlike those of many other crops, the sugarcane genome is highly heterozygous due to its autopolyploid nature, which further hinders the development of a comprehensive genetic map. Despite these limitations, many superior agronomic traits/genes for higher cane yield, sugar production, and disease/pest resistance have been identified through the mapping of quantitative trait loci, genome-wide association studies, and transcriptome approaches. Improvements in traits controlled by one or two loci are relatively easy to achieve; however, this is not the case for traits governed by many genes. Many desirable phenotypic traits are controlled by quantitative trait nucleotides (QTNs) with small and variable effects. Assembling these desired QTNs by conventional breeding methods is time consuming and inefficient due to genetic drift. However, recent developments in genomics selection (GS) have allowed sugarcane researchers to select and accumulate desirable alleles imparting superior traits as GS is based on genomic estimated breeding values, which substantially increases the selection efficiency and genetic gain in sugarcane breeding programs. Next-generation sequencing techniques coupled with genome-editing technologies have provided new vistas in harnessing the sugarcane genome to look for desirable agronomic traits such as erect canopy, leaf angle, prolonged greening, high biomass, deep root system, and the non-flowering nature of the crop. Many desirable cane-yielding traits, such as single cane weight, numbers of tillers, numbers of millable canes, as well as cane quality traits, such as sucrose and sugar yield, have been explored using these recent biotechnological tools. This review will focus on the recent advances in sugarcane genomics related to genetic gain and the identification of favorable alleles for superior agronomic traits for further utilization in sugarcane breeding programs.

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Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Cited by 44 publications

References 48 publications

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance

Genetic Transformation of Sugarcane, Current Status and Future Prospects

Recent Advances in Sugarcane Genomics, Physiology, and Phenomics for Superior Agronomic Traits

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