CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis

Abstract

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of systemwide losses of gene function.

Sorghum bicolorcultivars have divergent and dynamic gene regulatory networks that control the temporal expression of genes in stem tissue

“…However, further studies are needed to confirm “real” TF-target interactions through experiments such as yeast-one hybrid (Y1H) (Arda and Walhout, 2009; Fuxman Bass, Reece-Hoyes and Walhout, 2016) or DAP-seq (O’Malley et al , 2016). Nonetheless, the current study provides a starting point for more in-depth experiments to assess the extent to which these promoter elements are tissue-specific through targeted mutagenesis, for example, using tissue-specific genome modification (Schürholz et al , 2018; Decaestecker et al , 2019; Shi et al , 2019; Ali, Mahfouz and Mansoor, 2020). This will greatly benefit efforts to fine-tune manipulation of tissue-specific and developmental-stage specific gene expression of transgenes required to genetically engineer sorghum stems to accumulate oil and elevate its status as a biofuel crop.…”

Brassinosteroids: Multidimensional Regulators of Plant Growth, Development, and Stress Responses

“…Tissue-specific promoters, such as vascular tissue-specific S-ADENOSYLMETHIONINE SYNTHASE (Wu et al, 2008) and seedspecific glutelin Gt1 promoters , have been employed to drive the overexpression of BR biosynthetic genes, such as genes encoding sterol C-22 hydroxylases (which control the conversion of campestanol to 6-deoxocathasterone; Wu et al, 2008) and OsDWARF4 in rice to increase seed yield without impairing other traits. A CRISPR-based tissue-specific knockout system can be designed and used to generate mutations in particular cell types and tissues (Decaestecker et al, 2019). Applying both of these tissue-specific gene manipulation systems to fine-tune BR signaling as needed would facilitate the generation of improved crops.…”

Targeted genome editing inNicotiana tabacumusing inducible CRISPR/Cas9 system

“…However, constitutive expression of Cas9 is not necessary, given the fact that transient expression of Cas9 in plants is enough to induce the targeted mutagenesis [35,45,46]. Moreover, knockout of fundamentally significant genes (almost 10% of the Arabidopsis genome) would result in severe pleiotropic effects or lethality [47,48], and the lack of corresponding loss-of-function mutants makes it impossible to decipher the functions of these indispensable genes. More accurate editing would be preferred considering that many genes express in specific context in plants [48].…”

“…Moreover, knockout of fundamentally significant genes (almost 10% of the Arabidopsis genome) would result in severe pleiotropic effects or lethality [47,48], and the lack of corresponding loss-of-function mutants makes it impossible to decipher the functions of these indispensable genes. More accurate editing would be preferred considering that many genes express in specific context in plants [48]. Inducible genome editing can be used as an approach to overcome the limitations and accomplish temporal and spatial editing of genes of interest.…”