Abstract
Genome editing (also known as gene editing) employs a range of tools such as Meganucleases, Zinc Finger Nucleases, TALENs, and more recently CRISPR to make defined changes in genes, regulatory sequences, untranslated regions, or intergenic regions. It is increasingly being applied in plant science research and to improve plant varieties. The benefits of having effective detection tools begin with optimization of the genome editing process itself and continue with selection and characterization of tissue cultures and/or regenerated plants. Detection tools are also used throughout the breeding process, and for preparation of regulatory dossiers when required, as well as for seed production, and may be necessary for monitoring products in the marketplace. Detection and identification of genome edits employs a wide range of analytical approaches including PCR, digital PCR, and sequencing methods. This article examines the applicability of each category of detection or identification approach, from the optimization of genome editing processes, through creation of edits, selection and characterization, and breeding. The challenges surrounding the detection of genome edits present at low levels in large seed, plant, or grain populations and of differentiating directed genome edits from conventional mutations are also explained.
Highlights
Plant breeders have traditionally relied on selection of suitable genotypes from populations, or after wide crosses to leverage the naturally occurring variability in the genome
CRISPR nucleases or other effectors can be transiently expressed in plant cells or CRISPR reagents can be delivered in a DNA-free manner using ribonucleoproteins (RNPs) (Hamada et al 2018)
Many different types of detection tools are used in the field of genome editing
Summary
Plant breeders have traditionally relied on selection of suitable genotypes from populations, or after wide crosses to leverage the naturally occurring variability in the genome. The need for detection tools begins with optimization of the genome editing process itself and continues with characterization of tissue cultures and/or regenerated plants. Detection and identification of genome edits employ a wide range of analytical approaches throughout the development process. These include phenotypic characterization, PCR, digital PCR, and sequencing methods. Optimization of genome editing processes requires a range of analytical approaches as detection in tissue cultures can be challenging. Applications of detection tools to single plants at early stages of the genome editing process may not be as challenging and presence/absence or zygosity assays (via PCR or digital PCR) are sufficient for this purpose. This article will cover the detection tools used in optimization of genome editing and the early stages of research and in breeding. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas) SDN editing has enabled an increase in the application of genome editing tools to plant modification and breeding (Chen et al 2019)
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