Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology

Khan, Muhammad Hafeez Ullah; Khan, Shahid Ullah; Ali, Muhammad; Hu, Limin; Yang, Yang; Fan, Chuchuan

doi:10.1002/jcp.26299

Cited by 22 publications

(16 citation statements)

References 70 publications

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“…CRISPR/Cas9 has a number of advantages, including the generation of complete knockouts without necessarily transgenic sequences present in the final mutant. Nevertheless, this technique is rather laborious, especially when multiple alleles are to be targeted, may lead to off‐target effects and usually has a requirement for homozygosity (Khan Ullah et al , ). In addition, lethality may occur if the targeted gene is essential in specific developmental stages.…”

Section: Resultsmentioning

confidence: 99%

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

Κατσαρού

Mitta

Bardani

et al. 2018

Molecular Plant Pathology

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Summary RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer‐like (DCL), Argonaute (AGO) and RNA‐dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana , an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next‐generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.

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Section: Resultsmentioning

confidence: 99%

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

Κατσαρού

Mitta

Bardani

et al. 2018

Molecular Plant Pathology

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“…The canonical CRISPR/Cas9 RGEN system offers a variety of applications in plant biotechnology, from crop breeding to plant synthetic biology. The efficiency of SpCas9 in genome editing has been demonstrated in dozens of plant species, where it has been used to produce new traits of agronomic importance, including resistance to abiotic stress and herbicides (Khan et al, 2017;Soda et al, 2017;Yin et al, 2017a). Despite its success, further expansion of the CRISPR toolbox with new RGEN tools will be necessary to overcome inherent limitations of SpCas9.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

113

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Summary The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

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“…In recent years, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) system is considered the most simple and effective sequence-specific nuclease. It has been used for genome editing in major crops (Ahmar et al, 2020;M. H. U. Khan et al, 2018).…”

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confidence: 99%

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production

Khan

Zhu

et al. 2020

Journal Cellular Physiology

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Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T 1 and T 2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T 1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

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Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology

Cited by 22 publications

References 70 publications

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

DCL‐suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology

Assessment of Cas12a‐mediated gene editing efficiency in plants

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production

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