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Heritable Genome Editing in Plants
Tissue-Specific Gene Knockdown
Advantages and Applications of Tissue-Specific Gene Knockdown
Limitations of Tissue-Specific Gene Knockdown
References
Heritable Genome Editing in Plants The adaptive immune system of prokaryotes, [clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas system)] CRISPRCas9 system, has been domesticated as a powerful genome-editing tool that has revolutionized the functional genomics of eukaryotes [1]. In the Streptococcus pyogenes CRISPR/Cas9 system, Cas9 endonuclease is directed by a single guide RNA to a complementary 20-nucleotide target DNA sequence where, upon the recognition of the protospacer-associated motif (NGG), the active Cas9 make DNA double-stranded breaks (DSBs) [2] and the following imprecise repair of DSBs leads to targeted mutations in DNA. The CRISPR-Cas9 system has been established successfully in the majority of model and crop species for functional knockouts and trait improvements [3,4]. Similarly, coupling of the CRISPR-Cas9 system with a homologydirected repair system paved the way for precise genome engineering in plants [5]. Moreover, CRISPR-Cas9 was applied to produce foreign DNA-free, non-genetically modified organism (GMO)-improved crops [6]. However, all of these genome-editing approaches have two main objectives, that is, to improve the rate of targeted mutagenesis and to recover mutant progenies [1]. For that, various methodologies like use of regulatory elements (enhancers, promoters, and terminators), specific cell-cycle/phases, viral replicons, and physical and chemical conditions were applied to enhance expression of CRISPR-Cas9 machinery and hence the recovery of edited plants [3,4,7]. Despite their success, it remains that more than 10% of Arabidopsis (Arabidopsis thaliana) genes, cannot be characterized with these methods. These genes are essential or have pleiotropic effects and their complete removal interferes with the cellular machinery, reproduction, or development [1]. In the absence of heritable homozygous knockouts for these essential and pleiotropic genes, their functions are assumed and their real roles remain unknown.
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