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CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

First author: Ward Decaestecker; Affiliations: Ghent University (根特大学)Ghent, Belgium

Corresponding author: Thomas B. Jacobs

Detailed functional analyses of many fundamentally-important plant genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. 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 CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to discover and analyze gene functions in spatial and temporal contexts of plant life while avoiding pleiotropic effects of system-wide loss of gene function.



通讯:Thomas B. Jacobs (http://www.vib.be/en/research/scientists/Pages/Thomas-Jacobs-Lab.aspx)

个人简介:2014年,美国佐治亚大学,博士;2014-2016年,美国Boyce Thompson研究所,博士后。


doi: http://dx.doi.org/10.1101/474981

Journal: bioRxiv

First Posted: November 20, 2018

An inducible genome editing system for plants

First author: Xin Wang; Affiliations: University of Helsinki (赫尔辛基大学)Helsinki, Finland

Corresponding author: Ari Pekka Mähönen

Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a novel tool with which target genes can be efficiently conditionally knocked out at any developmental stage. The target gene is manipulated using the CRISPR-Cas9 genome editing technology, and conditionality is achieved with the well-established estrogen-inducible XVE system. Target genes can also be knocked-out in a cell-type specific manner. Our tool is easy to construct and will be particularly useful for studying genes which have null-alleles that are non-viable or show strong developmental defects.



通讯:Ari Pekka Mähönen (https://researchportal.helsinki.fi/en/persons/ari-pekka-mähönen)



doi: http://dx.doi.org/10.1101/779140

Journal: bioRxiv

First Posted: September 23, 2019

Live imaging-assisted domain-specific CRISPR genome editing at single cell resolution in plants

First author: Ting Li; Affiliations: Gregor Mendel Institute of Molecular Plant Biology (孟德尔分子植物生物学研究所)Vienna, Austria

Corresponding author: Elliot M. Meyerowitz

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has been widely used for genome engineering in a wide range of organisms, but much of the development of CRISPR-based genome editing has been aimed toward improving its efficiency and accuracy, so as to obtain genetic materials carrying known and stably heritable genome modifications. Precise spatiotemporal control over genome editing technology at cell type resolution is a key challenge for gene function studies. Some tissue-specific CRISPR genome editing methods relying on phenotypic characterization and fluorescent immune-staining techniques have been developed for biomedical research and gene therapy, they function by spatially controlling expression of Cas9. Recent work establishes the presence and location of mutational events at a single cell level in Arabidopsis roots and stomata. Here we present an efficient domain-specific CRISPR-Cas9 system combined with a high resolution live-imaging based screening strategy, applied in the shoot apical meristem of Arabidopsis thaliana. Using the system we investigate PIN-FORMED1 (PIN1) protein functions in tissue morphogenesis and PIN1 mechanical stress response in a cell layer-specific fashion. We find that reported failure to generate new primordia in epidermal PIN1 knockout SAMs is due to a reduction in mechanical stress differences in the sub-epidermal layer. The methods described are applicable to spatial-temporal gene manipulation in plants.



通讯:Elliot M. Meyerowitz (https://www.bbe.caltech.edu/people/elliot-meyerowitz)



doi: http://dx.doi.org/10.1101/793240

Journal: bioRxiv

First Published: October 04, 2019



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