Optimized prime editing efficiently generates heritable mutations in maize

Qiao, Dexin; Wang, Junya; Lu, Minhui; Xin, Cuiping; Chai, Yiping; Jiang, Yuanyuan; Sun, Wei; Cao, Zhenghong; Guo, Siyi; Wang, Xue-Chen; Chen, Qijun

doi:10.1111/jipb.13428

Cited by 23 publications

(20 citation statements)

References 19 publications

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“…In 2023, Qiao et al added a structured RNA motif, evopreQ1, to the 3′ end of pegRNAs to improve their stability by preventing degradation and named the motif epegRNAs. They also optimized the PE2 protein to obtain PEmax and over-expressed the MLH1 protein with a disrupted active structural domain (MLH1dn) to inhibit the DNA mismatch repair pathway, thereby substantially improving the efficiency of PE editing in the maize genome [ 109 ].…”

Section: Strategies and Methods For Optimizing Crispr/cas9 Gene-editi...mentioning

confidence: 99%

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Zhou

Luan

Liu

et al. 2023

Plants

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Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

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Section: Strategies and Methods For Optimizing Crispr/cas9 Gene-editi...mentioning

confidence: 99%

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Zhou

Luan

Liu

et al. 2023

Plants

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“…Prime editing (PE) is one of the CRISPR-Cas-based gene editing approaches that allow in vivo installation of multiple types of precise DNA changes with low off-targeting activities [2][3][4] . The PE technique has been successfully applied and improved in monocots [5][6][7][8][9][10][11][12][13] . However, the PE performance in dicots was extremely low and practically unsuccessful in the whole plant level [14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

“…Several strategies derived from animal and plant research have been proposed to address the inefficiency and inconsistency of PE. Notable methods include the PE2max configuration, ‘flip and extension’ pegRNA protection, RNase H-truncated RT variant, nucleocapsid RNA chaperone use, Primer Binding Site (PBS) design within an optimal Tm range, and composite promoter use for driving pegRNA transcription 8, 10, 12, 13, 15, 18 . Despite these advancements, the application of PE in dicots, specifically tomatoes, has been challenging due to efficiency issues potentially caused by factors such as self-complementarity of the PBS and spacer sequence, RTT complexity, PE protein stability, unfavorable reaction conditions, and pegRNA stability 4 .…”

Section: Introductionmentioning

confidence: 99%

Breakthrough in Dicot Prime Editing: Enabling Heritable Desired Edits in Tomato andArabidopsis

Vu,

Nguyen,

Kim

et al. 2024

Preprint

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Prime editing (PE) enables almost all types of precise genome editing in animals and plants. It has been successfully adapted to edit several plants at variable efficiency and versatility. However, this technique is inefficient for dicots for unknown reasons. Here, by employing novel combinations of PE components, including an RNA chaperone and modified epegRNAs driven by a PolII-PolIII composite promoter and a viral replicon system, we obtained up to 9.7% of the desired PE efficiency at the callus stage assessed by targeted deep sequencing. Subsequently, we identified that up to 38.2% of transformants contained desired PE alleles in tomatoes andArabidopsis, marking the first successful heritable PE transmission in dicots. Our PE tools also showed high accuracy, specificity, and multiplexing capability, which unlocked the potential for practical PE applications in dicots, paving the way for transformative advancements in plant sciences.

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“…The strategy of MMR suppression has also been attempted in plant PE engineering. For instance, compared with ePE3max, ePE5max expressing a dominant-negative variant of rice MLH1 ( OsMLH1dn ) or maize MLH1dn ( ZmMLH1dn ) increased the proportion of homozygous mutations in stable transgenic lines [ 13 , 14 ]. However, ePE5max using OsMLH1dn exhibited comparable but not significantly enhanced efficiency compared with ePE3max in protoplasts, and the same finding was obtained for the overall mutation efficiency in transgenic rice [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice

Liu,

Gu,

Zhang

et al. 2024

Genome Biol

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Background High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. Results We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. Conclusions This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

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Optimized prime editing efficiently generates heritable mutations in maize

Cited by 23 publications

References 19 publications

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Breakthrough in Dicot Prime Editing: Enabling Heritable Desired Edits in Tomato andArabidopsis

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice

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