TY - JOUR

T1 - Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants

AU - Hu, Yangjie

AU - Patra, Priyanka

AU - Pisanty, Odelia

AU - Shafir, Anat

AU - Belew, Zeinu Mussa

AU - Binenbaum, Jenia

AU - Ben Yaakov, Shir

AU - Shi, Bihai

AU - Charrier, Laurence

AU - Hyams, Gal

AU - Zhang, Yuqin

AU - Trabulsky, Maor

AU - Caldararu, Omer

AU - Weiss, Daniela

AU - Crocoll, Christoph

AU - Avni, Adi

AU - Vernoux, Teva

AU - Geisler, Markus

AU - Nour-Eldin, Hussam Hassan

AU - Mayrose, Itay

AU - Shani, Eilon

N1 - Publisher Copyright: © 2023, The Author(s), under exclusive licence to Springer Nature Limited.

PY - 2023

Y1 - 2023

N2 - Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat 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 that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs 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.

AB - Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat 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 that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs 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.

U2 - 10.1038/s41477-023-01374-4

DO - 10.1038/s41477-023-01374-4

M3 - Journal article

C2 - 36973414

AN - SCOPUS:85150958102

VL - 9

SP - 572

EP - 587

JO - Nature Plants

JF - Nature Plants

SN - 2055-026X

IS - 4

ER -