Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants
Résumé
Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions 1. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programs 2. Here, we describe the development and validation of Multi-Knock, a genome-scale CRISPR toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single guide RNAs (sgRNAs) that each target 2 to 10 genes within a family at once. Furthermore, partitioning the library into ten sub-libraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 sgRNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.
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