A modular cloning toolkit for genome editing in plants

Hahn, Florian; Korolev, Andrey V.; Loures, Laura Sanjurjo; Nekrasov, Vladimir

doi:10.1186/s12870-020-02388-2

Cited by 46 publications

(28 citation statements)

References 48 publications

(107 reference statements)

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“…In its current state, the pDGE system can handle up to 32 sgRNA units, but can be extended following the same design principles. Alternatively, highly complex nuclease constructs may also be assembled using the modular cloning system (Grützner et al , 2020; Hahn et al , 2019; Weber et al , 2011). Modular cloning offers maximal flexibility, while the pDGE system, based on pre‐assembled recipient vectors containing a range of different zCas9 expression cassettes and selection markers, provides simplicity of use.…”

Section: Discussionmentioning

confidence: 99%

“…Accordingly, RGNs are frequently used in the multiplexing mode (reviewed in Armario Najera et al , 2019), and comprehensive toolkits for assembly of respective nuclease‐coding constructs are available (e.g., Cermak et al , 2017; Hahn et al , 2019; Lowder et al , 2015; Wu et al , 2018; Xing et al , 2014). However, reduced efficiencies can be expected when an increasing number of sgRNAs is used for multiplexing, as individual sgRNAs will compete for the common nuclease core.…”

Section: Introductionmentioning

confidence: 99%

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Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

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SUMMARY Genome editing by RNA‐guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter‐selection markers for N. benthamiana, most importantly Sl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

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“…In its current state, the pDGE system can handle up to 32 sgRNA units, but can be extended following the same design principles. Alternatively, highly complex nuclease constructs may also be assembled using the Modular Cloning system (Weber et al , 2011, Hahn et al , 2019, Grützner et al , 2020). Modular Cloning offers maximal flexibility, while the pDGE system, based on preassembled recipient vectors containing a range of different zCas9 expression cassettes and selection markers, provides simplicity of use.…”

Section: Discussionmentioning

confidence: 99%

“…Accordingly, RGNs were frequently used in the multiplexing mode (reviewed in Armario Najera et al , 2019), and comprehensive toolkits for assembly of respective nuclease-coding constructs are available (e.g., Xing et al , 2014, Lowder et al , 2015, Cermak et al , 2017, Wu et al , 2018, Hahn et al , 2019). However, reduced efficiencies can be expected when an increasing number of sgRNAs is used for multiplexing, as individual sgRNAs will compete for the common nuclease core.…”

Section: Introductionmentioning

confidence: 99%

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

Stuttmann

Barthel

Martin

et al. 2020

Preprint

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SummaryGenome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.

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“…A variety of comprehensive, recent reviews have focused on advances in gene editing technologies (Razzaq et al 2019;Bilichak et al 2020;Gürel et al 2020;Hahn et al 2020;He and Zhao 2020;Hsieh-Feng and Yang 2020;Li and Xia 2020), and the application of these techniques to select crops such as rice (Oryza sativa, Biswal et al 2019), bread wheat (Triticum aestivum; Borisjuk et al 2019;Kumar et al 2019;Hensel 2020), maize (Zea mays; Agarwal et al 2018,), soybean (Glycine max; Bao et al 2020), sorghum (Sorghum bicolor; Char and Yang 2020), and the improvement of certain traits, such as abiotic stress tolerance (Abdelrahman et al 2018), disease resistance (Zaidi et al 2016;Borrelli et al 2018;Bisht et al 2019), and grain quality (Fiaz et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Gene editing applications to modulate crop flowering time and seed dormancy

et al. 2020

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Gene editing technologies such as CRISPR/Cas9 have been used to improve many agricultural traits, from disease resistance to grain quality. Now, emerging research has used CRISPR/Cas9 and other gene editing technologies to target plant reproduction, including major areas such as flowering time and seed dormancy. Traits related to these areas have important implications for agriculture, as manipulation of flowering time has multiple applications, including tailoring crops for regional adaptation and improving yield. Moreover, understanding seed dormancy will enable approaches to improve germination upon planting and prevent pre-harvest sprouting. Here, we summarize trends and recent advances in using gene editing to gain a better understanding of plant reproduction and apply the resulting information for crop improvement.

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A modular cloning toolkit for genome editing in plants

Cited by 46 publications

References 48 publications

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

Gene editing applications to modulate crop flowering time and seed dormancy

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