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 system-wide losses of gene function.
Highlights
The most 11" commonly used clustered regularly interspaced short palindromic repeat (CRISPR) system in plants is based on the CRISPR-associated 9 (Cas9) 12" DNA endonuclease and its artificial CRISPR RNAs (crRNA), the guide RNA (Jinek et al, 2012). 13" In plants, Cas9 is very efficient at inducing double-strand DNA breaks
54" Here, we present a method for tissue-specific genome modification via a CRISPR 55" tissue-specific knockout (CRISPR-TSKO) vector system in Arabidopsis that allows for
We demonstrate the potential of CRISPR-TSKO for somatic gene 59" knockouts of several essential genes in diverse plant cell types, tissues, and organs
Summary
2" The generation of stable, inheritable loss-of-function mutant alleles has been 3" indispensable for functional genomic studies in plants. 18" Most CRISPR efforts in plants to date have focused on generating stable and heritable 19" mutant alleles for reverse genetics approaches This approach is limited, as the 20" knockout of many fundamentally important genes convey severe pleiotropic phenotypes 21" or even lethality. The use of tissue-specific promoters to drive Cas expression have been 47" reported in plants (Hyun et al, 2015; Yan et al, 2015; Mao et al, 2016) These 48" efforts have focused on increasing the recovery of stably transmitted mutant alleles. 49" Recently, the fiber-specific NST3/SND1 promoter was used to drive Cas expression 50" and target the essential gene HCL (encoding a hydroxycinnamoyl transferase) in 51" Arabidopsis (Liang et al, 2019) This approach demonstrated that xylem-specific Cas9 52" expression is able to decrease lignin content in xylem cells while avoiding 53" the strong pleiotropic growth defects of full knockout mutants. Our approach opens opportunities to study the 62" functions of fundamentally important genes in specific contexts of plant development 63" and creates the possibility of investigating post-embryonic developmental processes
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