<scp>CRISPR</scp>‐S: an active interference element for a rapid and inexpensive selection of genome‐edited, transgene‐free rice plants

Lu, Haiping; Liu, Song-Mei; Xu, Shengbao; Chen, Wu-Yang; Zhou, Xin; Tan, Yuanyuan; Huang, Jianzhong; Shu, Qingyao

doi:10.1111/pbi.12788

Cited by 84 publications

(65 citation statements)

References 10 publications

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“…Recently, several strategies have been developed to accelerate the removal of transgene components while retaining the desired mutations. These strategies include the integration of fluorescent markers 112,113 , herbicide-dependent isolation system 114 , and the programmed self-elimination system 115 .…”

Section: Transgene-free Genome Editingmentioning

confidence: 99%

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Sapkota

Knaap

2020

Hortic Res

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Directed breeding of horticultural crops is essential for increasing yield, nutritional content, and consumer-valued characteristics such as shape and color of the produce. However, limited genetic diversity restricts the amount of crop improvement that can be achieved through conventional breeding approaches. Natural genetic changes in cisregulatory regions of genes play important roles in shaping phenotypic diversity by altering their expression. Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement through the introduction of genetic variation in a targeted manner. The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) provides a more refined method for modulating gene expression and creating phenotypic diversity to benefit crop improvement. Here, we focus on the current applications of CRISPR/Cas-mediated cis-engineering in horticultural crops. We describe strategies and limitations for its use in crop improvement, including de novo cis-regulatory element (CRE) discovery, precise genome editing, and transgene-free genome editing. In addition, we discuss the challenges and prospects regarding current technologies and achievements. CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural crops that are better able to adapt to climate change and providing food for an increasing world population.

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Section: Transgene-free Genome Editingmentioning

confidence: 99%

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Sapkota

Knaap

2020

Hortic Res

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“…Another reported strategy for isolating transgene-free edited plants was to couple the CRISPR construct with an RNA interference element, which targets a herbicideresistance P450 enzyme. Such a strategy enables herbicidedependent isolation of transgene-free plants (Lu et al, 2017). However, this strategy still does not enrich the transgene-free plants and requires planting the population.…”

mentioning

confidence: 99%

Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants

et al. 2018

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CRISPR gene-editing technology has successfully generated targeted mutations in rice and many other plant species (Ma et al., 2015). Assessment of heritability and phenotypic stability of CRISPR-edited plants requires the elimination of the CRISPR construct. The presence of the CRISPR construct makes it difficult to distinguish the mutations transmitted from the previous generation from newly generated mutations by the CRISPR construct at the current generation. The existence of the CRISPR construct also greatly increases the risk of off-target effects, making phenotypic stability a concern. Moreover, removal of the transgenes is likely a prerequisite for gaining regulatory approval of CRISPR-edited crops for commercial applications. Development of an efficient and easy-to-perform technology for the removal of the CRISPR construct after DNA modifications have been completed will have a major impact on using gene-editing technology for crop improvement.

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“…First, high-specificity targets should be carefully selected using in silico predictions (Haeussler et al, 2016). Second, Cas9-free mutants should be isolated as far as possible Lu et al, 2017). Third, when necessary, high-specificity SpCas9 variants in combination with the tRNA-sgRNA(m) fusion method can be used Kleinstiver et al, 2016;Kulcsar et al, 2017;Slaymaker et al, 2016;Xie et al, 2015;Zhang et al, 2017a).…”

Section: / 26mentioning

confidence: 99%

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention

Zhang

Huang

Wang

et al. 2017

Preprint

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Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score.We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

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CRISPR‐S: an active interference element for a rapid and inexpensive selection of genome‐edited, transgene‐free rice plants

Cited by 84 publications

References 10 publications

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention

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