Enhancing HR Frequency for Precise Genome Editing in Plants

Abstract

Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.

CRISPR/Cas9-Mediated Cytosine Base Editing Using an Improved Transformation Procedure in Melon (Cucumis melo L.)

“…The CRISPR/Cas9 system has been successfully applied in various model plants and crops to engineer plants with desired traits, primarily leveraging the NHEJ pathway [ 36 , 37 ]. Another approach for plant genome editing, utilizing the HDR pathway, aims to introduce precise and specific mutations at the target site, but the efficiency of HDR is often low [ 38 ]. Inducing precise point mutations using the HDR process requires a template DNA carrying the desired sequence.…”

Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation

“…GE, through the CRISPR/Cas technology, offers an alternative approach for generating the induced variation at the target sites in the genome’s regulatory or coding regions. Additionally, targeted generation of the double-strand breaks (DSBs) allows, for example, induced recombination at the predefined positions ( Chen et al, 2022 ), introgression of specific loci from the close relatives or other cultivars, and elimination of linkage drag ( Lazar et al, 2020 ). Until recently, the stable wheat transformation was relatively challenging due to its high variability and genotype-dependency, resulting in low transformation efficiency (1%–2%).…”

Diversity of Transgene Integration and Gene Editing Events in Wheat (T<em>riticum aestivum</em> L.) Transgenic Plants Generated Using Agrobacterium-Mediated Transformation

“…GE, through the CRISPR/Cas technology, offers an alternative approach for generating the induced variation at the target sites in the genome's 2 regulatory or coding regions. Additionally, targeted generation of the double-strand breaks (DSBs) allows, for example, induced recombination at the predefined positions [4], introgression of specific loci from the close relatives or other cultivars, and elimination of linkage drag [5]. Until recently, the stable wheat transformation was relatively challenging with high variability, genotype-dependent, resulting in low transformation efficiency (1-2%).…”