A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis

Feng, Zhengyan; Zhang, Zhengjing; Hua, Kai; Gao, Xifeng; Mao, Yanfei; Botella, José Ramón; Zhu, Jian‐Kang

doi:10.3390/ijms19123925

Cited by 46 publications

(49 citation statements)

References 33 publications

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“…485 100 plants per progeny). Only 55 T2 plants had gl2 edited alleles and all of them were 486 chimeric plants (Feng et al 2018). These data clearly show that, even focusing the 487 screening of T2 plants on progenies of pre-selected T1 plants, it is extremely unlikely to 488 randomly pick T2 plants that have been edited in one gene, much more so in two or 489 three genes of interest.…”

Section: Design Of An Effective Crispr/cas9 Co-editing Proxy Vector 258mentioning

confidence: 92%

Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

Vavrik

Danna

2020

G3 Genes|Genomes|Genetics

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24CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-25 function mutants in plants, and hence a valuable tool to study gene function. This is 26 mainly due to the easy reprogramming of Cas9 specificity using customizable small 27 non-coding RNAs, and to the possibility of editing several independent genes 28 simultaneously. Despite these advances, the identification of CRISPR-edited plants 29remains time and resource-intensive. Here, based on the premise that one editing event 30in one locus is a good predictor of editing event/s in other locus/loci, we developed a 31 CRISPR co-editing selection strategy that greatly facilitates the identification of 32 CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting 33 the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed 34 mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an 35 easy-to-detect visible phenotype that is unrelated to the expected phenotype of the 36 gene/s under study. We tested this strategy via assessing the frequency of co-editing of 37 three functionally unrelated proxy genes. We found that each proxy predicted the 38 occurrence of mutations in each surrogate gene with efficiencies ranging from 68% to 39

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Section: Design Of An Effective Crispr/cas9 Co-editing Proxy Vector 258mentioning

confidence: 92%

Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

Vavrik

Danna

2020

G3 Genes|Genomes|Genetics

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“…For Arabidopsis gene editing, the 35S promoter, however, is not recommended because 35S-driven expression has low activity during embryogenesis and in germ-line cells when using floral dip transformation. Thus, the 35S promoter generates more somatic mutations and fewer mutations in the reproductive tissue, making the mutations less inheritable (Feng et al, 2018). The Arabidopsis ubiquitin (AtUBQ10) promoter, which is highly expressed during embryogenesis, is a better choice.…”

Section: Optimization Of Promoters Used In the Crispr/cas9 Systemmentioning

confidence: 99%

CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function

Zhang

Showalter

2020

Front. Plant Sci.

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For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

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“…The advancement of the CRISPR/Cas9 genome editing system through vector optimization provides opportunities for the technology to be applied toward generating desirable traits to maximize the biomass yield and/or quality for the betterment of mankind and sustainable agriculture through tackling the prevailing production constraints (Xing et al 2014;Gao et al 2015;Ma et al 2015;Svitashev et al 2015;Wang et al 2015a;Zhang et al 2017). Despite the effective and successful application of the CRISPR/Cas9 technology in general, there are still persisting challenges related to the low mutation efficiency in Arabidopsis and M. truncatula as compared to the improvement attained in monocot species, like rice and maize (Gao et al 2015;Wang et al 2015b;Yan et al 2015;Eid et al 2016;Mao et al 2016;Zhang et al 2017Zhang et al , 2018Le Blanc et al 2017;Feng et al 2018;Pauwels et al 2018;Castel et al 2019). In addition, the non-reproducibility and/or inconsistency of mutation efficiency are also prevailing limitations in Arabidopsis and Medicago truncatula (Wang et al 2015b;Eid et al 2016;Mao et al 2016;Meng et al 2017;Curtin et al 2018;Feng et al 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Despite the effective and successful application of the CRISPR/Cas9 technology in general, there are still persisting challenges related to the low mutation efficiency in Arabidopsis and M. truncatula as compared to the improvement attained in monocot species, like rice and maize (Gao et al 2015;Wang et al 2015b;Yan et al 2015;Eid et al 2016;Mao et al 2016;Zhang et al 2017Zhang et al , 2018Le Blanc et al 2017;Feng et al 2018;Pauwels et al 2018;Castel et al 2019). In addition, the non-reproducibility and/or inconsistency of mutation efficiency are also prevailing limitations in Arabidopsis and Medicago truncatula (Wang et al 2015b;Eid et al 2016;Mao et al 2016;Meng et al 2017;Curtin et al 2018;Feng et al 2018). The mutation efficiency of the CRISPR/Cas9 system is affected by multiple factors, including the expression level of Cas9-gRNA, the gRNA sequence, promoters driving Cas9 and small guide RNA (sgRNA), terminators, the composition of target sequence (spacer), T-DNA architecture, chromatin state, and the time frame of culture incubation (Mikami et al 2016;Gao et al 2018;Castel et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula

Wolabu

Park

Miao

et al. 2020

Planta

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Main conclusion An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. Abstract CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and monoallelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.

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A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis

Cited by 46 publications

References 33 publications

Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function

Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula

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