Precise genome modification in tomato using an improved prime editing system

Lu, Yuming; Tian, Yugang; Shen, Rundong; Yao, Qi; Zhong, Dating; Zhang, Xuening; Zhu, Jian‐Kang

doi:10.1111/pbi.13497

Cited by 109 publications

(80 citation statements)

References 10 publications

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“…Precise genome editing can be achieved through the homology-directed repair (HDR) pathway by introducing repair templates. Cas proteins can be further engineered to recruit effector protein domains in order to achieve base editing ( Kim, 2018 ), prime editing ( Li et al., 2020b ; Butt et al., 2020 ; Lin et al., 2020 ; Lu et al., 2020 ; Tang et al., 2020 ; Xu et al., 2020 ), epigenome editing ( Gallego-Bartolomé et al., 2018 ; Papikian et al., 2019 ), and transcriptional regulation ( Lowder et al., 2015 , 2018 ; Piatek et al., 2015 ; Li et al., 2017 ; Tang et al., 2017 ). To date, three major CRISPR systems for plant genome editing have been successfully demonstrated: Cas9, Cas12a, and Cas12b ( Zhang et al., 2019 ; Ming et al., 2020 ).…”

Section: Introductionmentioning

confidence: 99%

CRISPR ribonucleoprotein-mediated genetic engineering in plants

Zhang

Iaffaldano

2021

Plant Communications

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CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement. Commonly used Agrobacterium - or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis. Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site. Genetic engineering using CRISPR ribonucleoproteins (RNPs) has become an attractive approach with many advantages: DNA/transgene-free editing, minimal off-target effects, and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency. Although RNP-mediated genetic engineering has been demonstrated in many plant species, its editing efficiency remains modest, and its application in many species is limited by difficulties in plant regeneration and selection. In this review, we summarize current developments and challenges in RNP-mediated genetic engineering of plants and provide future research directions to broaden the use of this technology.

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Section: Introductionmentioning

confidence: 99%

CRISPR ribonucleoprotein-mediated genetic engineering in plants

Zhang

Iaffaldano

2021

Plant Communications

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“…The failure of the Cas9-RT/pegRNA complexes to produce indel mutations at all four loci might have resulted from the combined negative impacts of both components. Based on these data, we hypothesize that the activity of nCas9-RT/pegRNA complexes was affected by the negative impacts of both RT fusion and pegRNA structure, which led to extremely low efficiency, and by some unknown factor, resulting in especially low efficiencies in tomato and other dicot plants 13,14 . If the above assumption is true, then we expect that when the PBS is shortened, the PE efficiency will be improved.…”

Section: Resultsmentioning

confidence: 96%

“…1). That is also why Lu and coworkers could not obtain a significant improvement in PE activity when they incubated explants at high temperatures 14 . Thus, we hypothesize that a range of optimized melting temperature exists for an active PBS of pegRNA.…”

Section: Resultsmentioning

confidence: 99%

“…However, the majority of the data showed successful PE at the acetolactate synthase (ALS) locus. Very limited data regarding the applications of PE in dicots were released, with relatively low efficacy in potato 13 and tomato 14 , and this process would need to be improved for further applications. Therefore, we sought to investigate the activity of PE complexes in plants using a transient assay with a synthetic substrate in tobacco and stable transformation for editing ten loci in tomato.…”

Section: Introductionmentioning

confidence: 99%

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The obstacles and potential clues of prime editing applications in tomato, a dicot plant

Kim

Das

et al. 2021

Preprint

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Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a priming extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. However, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. However, our data obtained by targeted deep sequencing also revealed inefficient PE activity in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. These data could also help explain the recent successes of some prime editors in plants using improved expression systems. Our work provides an important clue for the application of the PE approach in crop improvement.

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“…A number of independent studies has demonstrated that PE is able to achieve genome modifications in plant protoplasts as well as stable lines, but with low efficiency. These studies were mostly performed in rice but also in tomato, potato and maize (Butt et al 2020;Hua et al 2020a;Jiang et al 2020;Li et al 2020b;Lin et al 2020;Lu et al 2020a;Tang et al 2020;Veillet et al 2020a;Xu et al 2020). Obviously, a couple of different factors might influence the editing efficiency in plants, such as the nature of the reverse transcriptase, thermal conditions, length of the template, length of PBS and the requirement of the second nick.…”

Section: Prime Editing: Critical Evaluationmentioning

confidence: 99%

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Huang

Puchta

2021

Transgenic Res

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In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

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Precise genome modification in tomato using an improved prime editing system

Cited by 109 publications

References 10 publications

CRISPR ribonucleoprotein-mediated genetic engineering in plants

CRISPR ribonucleoprotein-mediated genetic engineering in plants

The obstacles and potential clues of prime editing applications in tomato, a dicot plant

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

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