CRISPR/Cas brings plant biology and breeding into the fast lane

Schindele, Angelina; Dorn, A.; Puchta, Holger

doi:10.1016/j.copbio.2019.08.006

Cited by 91 publications

(50 citation statements)

References 54 publications

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“…to site-specific insertions and deletions that cannot be predefined like GT modifications (Schindele, Dorn, & Puchta, 2020). In the case of GT, the integration of exactly defined modifications is feasible by using a donor sequence for HR containing the desired modification flanked by homologous regions to the target locus.…”

Section: Introductionmentioning

confidence: 99%

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

2020

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CRISPR/Cas systems enable gene editing through the induction of site‐specific DNA double‐strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non‐homologous end joining (NHEJ)‐mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T‐rich regions. Most recently, we tested an improved, temperature‐tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants

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Section: Introductionmentioning

confidence: 99%

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

2020

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“…Targeted gene insertion at double-strand breaks (DSBs) in the GSHs provides a desirable alternative to conventional plant transformation methods 3 . Recent advances in genome editing technologies have enabled the induction of DSB at defined targets in a relatively simple manner, paving the way for targeted gene insertion in plants 4,5 .…”

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confidence: 99%

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

et al. 2020

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Targeted insertion of transgenes at predetermined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

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“…Traditionally, the domestication of plants and the development of productive varieties required decades of breeding, which is also the main reason why most breeding programs during the last 100 years concentrated on the further improvement of a relatively small number of crops that were domesticated already several thousand years ago. The recent discovery of multiple key domestication genes and scientific breakthroughs in introducing multiple genomic changes in plants simultaneously with CRISPR/Cas enables the domestication of wild species within a single plant generation (Schindele, Dorn, and Puchta 2019). De novo domestication can contribute to enhancing agrobiodiversity and dietary diversity with possible benefits for the environment and human nutrition (Singh et al 2019).…”

Section: New Domestication and Crop Diversitymentioning

confidence: 99%

Role of New Plant Breeding Technologies for Food Security and Sustainable Agricultural Development

Qaim

2020

Applied Eco Perspectives Pol

321

197

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New plant breeding technologies (NPBTs), including genetically modified and gene-edited crops, offer large potentials for sustainable agricultural development and food security while addressing shortcomings of the Green Revolution. This article reviews potentials, risks, and actually observed impacts of NPBTs. Regulatory aspects are also discussed. While the science is exciting and some clear benefits are already observable, overregulation and public misperceptions may obstruct efficient development and use of NPBTs. Overregulation is particularly observed in Europe, but also affects developing countries in Africa and Asia, which could benefit the most from NPBTs. Regulatory reforms and a more science-based public debate are required.

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CRISPR/Cas brings plant biology and breeding into the fast lane

Cited by 91 publications

References 54 publications

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

Using CRISPR/ttLbCas12a for in planta Gene Targeting in A. thaliana

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

Role of New Plant Breeding Technologies for Food Security and Sustainable Agricultural Development

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