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The Plant Genome Abstract - Original Research

Achieving Plant CRISPR Targeting that Limits Off-Target Effects

 

This article in TPG

  1. Vol. 9 No. 3
    unlockOPEN ACCESS
     
    Received: May 20, 2016
    Accepted: Aug 13, 2016
    Published: September 29, 2016


    * Corresponding author(s): jdwolt@iastate.edu
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doi:10.3835/plantgenome2016.05.0047
  1. Jeffrey D. Wolt *a,
  2. Kan Wangb,
  3. Dipali Sashitalc and
  4. Carolyn J. Lawrence-Dilld
  1. a Dep. of Agronomy and Biosafety Institute for Genetically Modified Agricultural Products
    b Dep. of Agronomy
    c Roy J. Carver Dep. of Biochemistry, Biophysics & Molecular Biology
    d Dep. of Genetics, Development, and Cell Biology and Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
Core Ideas:
  • Plant CRISPR-Cas9 genome editing may generate unintended off-target mutation.
  • Potential for off-target mutation is an important regulatory question for genome-edited plants.
  • Validated design approaches to discriminate target and potential off-target edits are needed.

Abstract

The CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein) has been used to generate targeted changes for direct modification of endogenous genes in an increasing number of plant species; but development of plant genome editing has not yet fully considered potential off-target mismatches that may lead to unintended changes within the genome. Assessing the specificity of CRISPR-Cas9 for increasing editing efficiency as well as the potential for unanticipated downstream effects from off-target mutations is an important regulatory consideration for agricultural applications. Increasing genome-editing specificity entails developing improved design methods that better predict the prevalence of off-target mutations as a function of genome composition and design of the engineered ribonucleoprotein (RNP). Early results from CRISPR-Cas9 genome editing in plant systems indicate that the incidence of off-target mutation frequencies is quite low; however, by analyzing CRISPR-edited plant lines and improving both computational tools and reagent design, it may be possible to further decrease unanticipated effects at potential mismatch sites within the genome. This will provide assurance that CRISPR-Cas9 reagents can be designed and targeted with a high degree of specificity. Improved and experimentally validated design tools for discriminating target and potential off-target positions that incorporate consideration of the designed nuclease fidelity and selectivity will help to increase confidence for regulatory decision making for genome-edited plants.

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