“…1 ). 37 Repair by NHEJ is often seamless, however on occasion errors occur leading to indels in DNA sequence that can disrupt gene function. 32 Alternatively, the HDR repair pathway allows for the integration of an entire gene if a donor template with complementary ends to the DSB break is co-delivered with the SSN (knock-in mutant).…”
Section: Methods Of Genetic Modification In Plantsmentioning
confidence: 99%
“… 32 Alternatively, the HDR repair pathway allows for the integration of an entire gene if a donor template with complementary ends to the DSB break is co-delivered with the SSN (knock-in mutant). 37 Figure 1. The repair pathways for double-stranded cuts in DNA.…”
Section: Methods Of Genetic Modification In Plantsmentioning
confidence: 99%
“…These reagents do not require DSB or utilize the DNA repair mechanism to generate the modifications. 37 The BE allows replacement of a single nucleotide base without requirement for a DSB or DNA donor template. This is achieved by modifying the Cas9 protein in two different ways.…”
Section: Future Perspectives In Potato Gene Editingmentioning
Potato (
Solanum tuberosum
L.) is the third most important crop worldwide and a staple food for many people worldwide. Genetically, it poses many challenges for traditional breeding due to its autotetraploid nature and its tendency toward inbreeding depression. Breeding programs have focused on productivity, nutritional quality, and disease resistance. Some of these traits exist in wild potato relatives but their introgression into elite cultivars can take many years and, for traits such as pest resistance, their effect is often short-lasting. These problems can be addressed by genetic modification (GM) or gene editing (GE) and open a wide horizon for potato crop improvement. Current genetically modified and gene edited varieties include those with Colorado potato beetle and late blight resistance, reduction in acrylamide, and modified starch content. RNAi hairpin technology can be used to silence the haplo-alleles of multiple genes simultaneously, whereas optimization of newer gene editing technologies such as base and prime editing will facilitate the routine generation of advanced edits across the genome. These technologies will likely gain further relevance as increased target specificity and decreased off-target effects are demonstrated. In this Review, we discuss recent work related to these technologies in potato improvement.
“…1 ). 37 Repair by NHEJ is often seamless, however on occasion errors occur leading to indels in DNA sequence that can disrupt gene function. 32 Alternatively, the HDR repair pathway allows for the integration of an entire gene if a donor template with complementary ends to the DSB break is co-delivered with the SSN (knock-in mutant).…”
Section: Methods Of Genetic Modification In Plantsmentioning
confidence: 99%
“… 32 Alternatively, the HDR repair pathway allows for the integration of an entire gene if a donor template with complementary ends to the DSB break is co-delivered with the SSN (knock-in mutant). 37 Figure 1. The repair pathways for double-stranded cuts in DNA.…”
Section: Methods Of Genetic Modification In Plantsmentioning
confidence: 99%
“…These reagents do not require DSB or utilize the DNA repair mechanism to generate the modifications. 37 The BE allows replacement of a single nucleotide base without requirement for a DSB or DNA donor template. This is achieved by modifying the Cas9 protein in two different ways.…”
Section: Future Perspectives In Potato Gene Editingmentioning
Potato (
Solanum tuberosum
L.) is the third most important crop worldwide and a staple food for many people worldwide. Genetically, it poses many challenges for traditional breeding due to its autotetraploid nature and its tendency toward inbreeding depression. Breeding programs have focused on productivity, nutritional quality, and disease resistance. Some of these traits exist in wild potato relatives but their introgression into elite cultivars can take many years and, for traits such as pest resistance, their effect is often short-lasting. These problems can be addressed by genetic modification (GM) or gene editing (GE) and open a wide horizon for potato crop improvement. Current genetically modified and gene edited varieties include those with Colorado potato beetle and late blight resistance, reduction in acrylamide, and modified starch content. RNAi hairpin technology can be used to silence the haplo-alleles of multiple genes simultaneously, whereas optimization of newer gene editing technologies such as base and prime editing will facilitate the routine generation of advanced edits across the genome. These technologies will likely gain further relevance as increased target specificity and decreased off-target effects are demonstrated. In this Review, we discuss recent work related to these technologies in potato improvement.
“…Several strategies that favor expression of the HDR pathway versus NHEJ repair have been reviewed recently [ 152 ]. Improving the availability of the donor repair template (DRT) by making it more accessible to the cell repair machinery and/or by increasing its copy number in the target cells has been tested in planta.…”
Section: Functional Gene and Allele Validation Approachesmentioning
confidence: 99%
“…Moreover, modified DRT architectures, such as the length and ratio of the homologous and non-homologous parts of the DRT, type and strandedness of the nucleic acid, presence of chemical modifications, and positioning of the DRT relative to the DSB, are also important elements for consideration in gene targeting experiments. Other approaches that have been considered are the utilization of alternative DSB inducers for increased efficiency of target cleavage and subsequently enhanced HDR, or the modulation of the DNA repair pathway to favor HDR by inactivating key genes promoting NHEJ [ 152 ].…”
Section: Functional Gene and Allele Validation Approachesmentioning
Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.
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