<i>GLABRA2</i>-based selection efficiently enriches Cas9-generated nonchimeric mutants in the T1 generation

Kong, Xiangjiu; Pan, Wenbo; Sun, Nengxu; Zhang, Tingyu; Liu, Lijing; Zhang, Huawei

doi:10.1093/plphys/kiab356

Cited by 20 publications

(17 citation statements)

References 40 publications

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“…In the plant genome editing processes, the cells with higher expression levels of Cas9 and sgRNA, which leads to higher mutation rates, are the desired ones for regeneration. Direct enrichment of these cells or plants could also increase the mutation efficiency, such as the GLABRA2 mutation-based visible selection (GBVS) system which adds the GL2 target as a visible selection marker to identify plants with high mutation efficiency ( Kong et al, 2021 ). However, mutation of a second gene might be a concern for crop breeding.…”

Section: Resultsmentioning

confidence: 99%

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Pan

Li²,

Li³

et al. 2022

Front. Plant Sci.

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The efficiency of the CRISPR/Cas9 genome editing system remains limited in many crops. Utilizing strong promoters to boost the expression level of Cas9 are commonly used to improve the editing efficiency. However, these strategies also increase the risk of off-target mutation. Here, we developed a new strategy to utilize intron-mediated enhancement (IME)-assisted 35S promoter to drive Cas9 and sgRNA in a single transcript, which escalates the editing efficiency by moderately enhancing the expression of both Cas9 and sgRNA. In addition, we developed another strategy to enrich cells highly expressing Cas9/sgRNA by co-expressing the developmental regulator gene GRF5, which has been proved to ameliorate the transformation efficiency, and the transgenic plants from these cells also exhibited enhanced editing efficiency. This system elevated the genome editing efficiency from 14–28% to 54–81% on three targets tested in lettuce (Lactuca sativa) without increasing the off-target editing efficiency. Thus, we established a new genome editing system with highly improved on-target editing efficiency and without obvious increasement in off-target effects, which can be used to characterize genes of interest in lettuce and other crops.

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Section: Resultsmentioning

confidence: 99%

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Pan

Li²,

Li³

et al. 2022

Front. Plant Sci.

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“…We edited AtBSD5 gene with Cas9, and we randomly sequenced four resistant plants and found two mutants. Cas9 gene expression was driven by the egg cell-specific promoter compared with 35S promoter in Arabidopsis , and the efficiency of gene editing was improved [ 24 , 25 ]. However, by screening seeds infected with AtBRI1 vector of sgRNA3, two resistant seedlings were obtained, one of which had target site mutation and produced the expected phenotype.…”

Section: Discussionmentioning

confidence: 99%

Application of CRISPR/CasΦ2 System for Genome Editing in Plants

Cai

Guo

Cao

et al. 2022

IJMS

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CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.

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“…CRISPR/Cas9 system-mediated genome editing leads to efficient target modification in plants, including the model plant Arabidopsis and several crop species ( Chen et al, 2019 ; Kong et al, 2021 ). This technology thus promises to accelerate basic research and crop improvement.…”

Section: Discussionmentioning

confidence: 99%

Transgene-free Genome Editing in Plants

Liu

Zhang

2021

Front. Genome Ed.

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Genome editing is widely used across plant species to generate and study the impact of functional mutations in crop improvement. However, transgene integration in plant genomes raises important legislative concerns regarding genetically modified organisms. Several strategies have been developed to remove or prevent the integration of gene editor constructs, which can be divided into three major categories: 1) elimination of transgenic sequences via genetic segregation; 2) transient editor expression from DNA vectors; and 3) DNA-independent editor delivery, including RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs). Here, we summarize the main strategies employed to date and discuss the advantages and disadvantages of using these different tools. We hope that our work can provide important information concerning the value of alternative genome editing strategies to advance crop breeding.

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GLABRA2-based selection efficiently enriches Cas9-generated nonchimeric mutants in the T1 generation

Cited by 20 publications

References 40 publications

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Application of CRISPR/CasΦ2 System for Genome Editing in Plants

Transgene-free Genome Editing in Plants

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