Low‐gluten, nontransgenic wheat engineered with CRISPR/Cas9

Sánchez-León, Susana; Gil-Humanes, Javier; Ozuna, Carmen V.; Giménez, María J.; Sousa, Carolina; Voytas, Daniel F.; Barro, Francisco

doi:10.1111/pbi.12837

Cited by 461 publications

(325 citation statements)

References 20 publications

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“…Thus, sequencing is useful for characterizing gluten genes to improve baking quality. Knowledge of gluten gene sequences may also be helpful in efforts to make wheat that people who suffer from celiac disease can eat, through gene editing (Sánchez‐León et al ).…”

Section: Yield and Quality Genesmentioning

confidence: 99%

Domestication and crop evolution of wheat and barley: Genes, genomics, and future directions

Haas

Schreiber

Mascher

2019

JIPB

101

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Wheat and barley are two of the founder crops of the agricultural revolution that took place 10,000 years ago in the Fertile Crescent and both crops remain among the world's most important crops. Domestication of these crops from their wild ancestors required the evolution of traits useful to humans, rather than survival in their natural environment. Of these traits, grain retention and threshability, yield improvement, changes to photoperiod sensitivity and nutritional value are most pronounced between wild and domesticated forms. Knowledge about the geographical origins of these crops and the genes responsible for domestication traits largely pre‐dates the era of next‐generation sequencing, although sequencing will lead to new insights. Molecular markers were initially used to calculate distance (relatedness), genetic diversity and to generate genetic maps which were useful in cloning major domestication genes. Both crops are characterized by large, complex genomes which were long thought to be beyond the scope of whole‐genome sequencing. However, advances in sequencing technologies have improved the state of genomic resources for both wheat and barley. The availability of reference genomes for wheat and some of its progenitors, as well as for barley, sets the stage for answering unresolved questions in domestication genomics of wheat and barley.

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Section: Yield and Quality Genesmentioning

confidence: 99%

Domestication and crop evolution of wheat and barley: Genes, genomics, and future directions

Haas

Schreiber

Mascher

2019

JIPB

101

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“…A comparatively challenging approach was taken by Sánchez‐León et al (), who simultaneously knocked out as many as 35 of the α‐gliadin genes of bread wheat, leading to low gluten content of grains with much reduced immunoreactivity. The fatty acid composition has a significant impact on human health.…”

Section: Applications Of Targeted Genome Modification By Nhejmentioning

confidence: 99%

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Kumlehn

Pietralla

Hensel

et al. 2018

JIPB

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Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)‐associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double‐strand break induction, resulting in mutations by non‐homologous recombination. Strategies for performing such experiments − from the design of guide RNA to the use of different transformation technologies − are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease‐deficient Cas9/12 proteins, as DNA‐binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

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“…In this respect, the CRISPR technology is expected to contribute greatly to creating novel variations in wheat. Wheat was one of the plant species first modified by CRISPR/Cas9 (Shan et al ., ), and several traits have been targeted for mutations, including powdery mildew resistance (Wang et al ., ), grain size (Wang et al ., ; Zhang et al ., ), and grain quality (Liang et al ., ; Sánchez‐León et al ., ; Zhang et al ., ). In all these researches, the Cas9 and sgRNA transgenes were delivered by the biolistic bombardment.…”

Section: Introductionmentioning

confidence: 99%

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

Zhang

Hua

Gupta

et al. 2019

Plant Biotechnology Journal

157

100

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Summary CRISPR /Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR /Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR /Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T 0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium ‐delivered CRISPR /Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR /Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, Ta CKX 2‐1 , Ta GLW 7 , Ta GW 2, and Ta GW 8 , in T 0 , T 1 , and T 2 generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in Ta CKX 2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium ‐delivered CRISPR /Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR /Cas9 remains active for novel mutations through generations.

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Low‐gluten, nontransgenic wheat engineered with CRISPR/Cas9

Cited by 461 publications

References 20 publications

Domestication and crop evolution of wheat and barley: Genes, genomics, and future directions

Domestication and crop evolution of wheat and barley: Genes, genomics, and future directions

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

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