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Research ArticleBREAKTHROUGH TECHNOLOGIES
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Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries

Thomas B. Jacobs, Ning Zhang, Dhruv Patel, Gregory B. Martin
Thomas B. Jacobs
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853
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  • ORCID record for Thomas B. Jacobs
  • For correspondence: thomas.jacobs@ugent.vib.begbm7@cornell.edu
Ning Zhang
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853
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Dhruv Patel
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853
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Gregory B. Martin
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853
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  • For correspondence: thomas.jacobs@ugent.vib.begbm7@cornell.edu

Published August 2017. DOI: https://doi.org/10.1104/pp.17.00489

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  • © 2017 American Society of Plant Biologists. All Rights Reserved.

Abstract

The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.

  • Glossary

    CRISPR
    clustered regularly interspaced short palindromic repeats
    gRNA
    guide RNA
    PAM
    protospacer adjacent motif
    DSB
    double-stranded break
    PTI
    pattern-triggered immunity
    ROS
    reactive oxygen species
    SMRT
    single molecule, real time
    CCS
    circular consensus sequences
    CFU
    colony-forming units
    • Received April 7, 2017.
    • Accepted June 17, 2017.
    • Published June 23, 2017.
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    Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries
    Thomas B. Jacobs, Ning Zhang, Dhruv Patel, Gregory B. Martin
    Plant Physiology Aug 2017, 174 (4) 2023-2037; DOI: 10.1104/pp.17.00489

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    Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries
    Thomas B. Jacobs, Ning Zhang, Dhruv Patel, Gregory B. Martin
    Plant Physiology Aug 2017, 174 (4) 2023-2037; DOI: 10.1104/pp.17.00489
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    Plant Physiology: 174 (4)
    Plant Physiology
    Vol. 174, Issue 4
    Aug 2017
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