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Table 2 Potential approaches for improvement of HGT in monocots shown in this review

From: Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

No.CategoryApproachHGT modeTested in organismHGT enhancement propertiesReferences
1homologous repair templateHomologous size and formExtrachromosomalNicotiana tabacumThe longer the better; Linearized forms were much betterBaur et al. 1990
2homologous repair templateHomologous size and formExtrachromosomalNicotiana plumbaginifoliaHomologous size was preferred to be more than 456 bpPuchta and Hohn 1991
3homologous repair templateHomologous formExtrachromosomalNicotiana tabacumssDNA was more preferredBilang et al. 1992; de Groot et al. 1992
4HDR-related genesEcRecA overexpressionIntrachromosomalNicotiana tabacum10 folds-efficiency increase with nuclear localized EcRECAReiss et al. 1996
5HDR-related genesRuvC overexpressionSomatic crossover; Extrachromosomal; intrachromosomalNicotiana tabacumSomatic crossover (12 folds); intrachromosomal recombination (11 folds); and extrachromosomal recombination (56 folds)Shalev et al. 1999
6HDR-related genesyeast RAD54 overexpressionSDSAArabidopsis thaliana27-fold enhancementShaked et al. 2005
7NHEJ and HDR-related genesRAD50 knockoutIntrachromosomalArabidopsis thaliana8–10 folds of SSA or intrachromosomal recombinationGherbi et al. 2001
8NHEJ related genesAtMLH1 knockoutIntrachromosomalArabidopsis thalianaKnockout mutation reduced 72% HDR frequenciesDion et al. 2007
9Chromatin modelingAtFAS1 or AtFAS2 knockoutInterchromosomalArabidopsis thaliana40-fold enhancement of somatic homologous recombinationEndo et al. 2006
10Cell Cycle Synchronization/S phaseHydroxyurea treatmentSDSAYarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris1.2- to 8-fold enhancementTsakraklides et al. 2015
11Cell Cycle Synchronization/S-G2 phasesCas9 fused with N-terminal (110a.a) of human GeminiSDSAHuman cell lines (HEK293T)72% enhancement of somatic homologous recombinationGutschner et al. 2016
12Culture conditionsPolyamines (putrescine, spermidine and spermine)SDSAMouse hair follicle model and Human cell lines (U2OS and HEK293)Polyamines promoted RAD51 loading onto ssDNAs in in vitro assaysLee et al. 2019
13NHEJ related genesSuppression by chemical inhibitorsSDSAHuman cell lines (HEK293T)KU70 and DNA ligase IV suppression by Scr7 obtained 4–5-fold increase of HR efficiencyChu et al. 2015
14NHEJ related genesSuppression by chemical inhibitorsSDSAHuman cell lines and mouseDNA ligase IV suppression by Scr7 enhanced CRISPR/Cas9-based HDR frequency up to 19 foldsMaruyama et al. 2015