Detection of genome edits in plants—from editing to seed

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.

Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration

“…In typical transgene-based selection, a chemical such as kanamycin is used to kill tissue without the kanamycin resistance transgene. In an edit-based approach, a chemical, typically an herbicide, is used to kill un-edited tissue, while an edit provides herbicide tolerance ( Shillito et al, 2021 ). Certain forms of herbicide tolerance require a particular amino acid substitution, making them more adaptable to edit-based selection.…”

“…Without selection pressure to reduce the background, this method is labor-intensive and requires high-throughput screening. To make this method feasible and cost-effective, researchers typically employ methods such as pooling samples, and screening these pools using sensitive methods, such as amplicon sequencing by Illumina ( Liu et al, 2019 ), high-resolution melt ( Chen et al, 2018 ), fragment analysis, or digital PCR ( Peng et al, 2020 ), reviewed in ( Shillito et al, 2021 ). A few exceptional successes (>3% efficiency) have been reported in wheat and maize using entirely no selection following particle bombardment with ribonucleoproteins ( Dong et al, 2021 ; Poddar et al, 2023 ; Liang et al, 2017 ).…”

CRISPR/Cas9-gene editing approaches in plant breeding

“… 80 Meanwhile, PCR assay-based approaches such as Kompetitative allele-specific PCR (KSAP), annealing at critical temperature PCR (ACT-PCR), allele-specific oligonucleotide PCR (ASO), and restriction fragment length polymorphism PCR (RFLP-PCR) have also been reported in number of known plant species. 81 However, many of the approaches mentioned above have limitations, and as they are time consuming and tedious, these cannot be used as a frontline testing tactic. To circumvent these troubleshooting, various revolutionized changes may have been made in conventional plant breeding methods over time that has an economic importance and save time to increase the speed of breeding that can usually take up to 10 years for variety development.…”

Selection and evaluation of gene-edited knockout mutants of AtAAP2 and AtCRF4 homologs of rice for agronomic nitrogen use efficiency (ANUE)

“…However, detecting the genetic differences between the original KO and segregant lines can be challenging since they may be due to somaclonal or off-target mutations, although our gRNAs were designed to reduce the possibility of this unfavorable event (Miyao et al, 2012;Zhang et al, 2015). Detecting genetic mutations in non-target regions is usually more laborious than sequencing the target regions (Shillito et al, 2021).…”