A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants

Tang, Xu; Zheng, Xuelian; Qi, Yiping; Zhang, Dengwei; Cheng, Yan; Tang, Aiting; Voytas, Daniel F.; Zhang, Yong

doi:10.1016/j.molp.2016.05.001

Cited by 152 publications

(140 citation statements)

References 11 publications

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“…Although we have previously shown that gRNA transcripts processed by ribozymes efficiently induce mutagenesis in rice, tobacco, and Arabidopsis (Tang et al, 2016), of the polycistronic RNA processing mechanisms tested in this study, ribozymes resulted in the lowest mutation frequencies. One difference between the two studies is that we expressed Cas9 and the gRNA array from separate promoters, whereas Tang et al (2016) used a single transcription unit to express both the Cas9 and the gRNAs.…”

Section: Discussioncontrasting

confidence: 59%

“…Processing polycistronic transcripts containing multiple gRNAs provides an alternative to the use of independent Pol III promoters. The polycistronic message can be produced from a Pol II promoter that is processed either by the Csy4 protein, tRNA processing enzymes, or ribozymes (Nissim et al, 2014;Aeby et al, 2010;Tang et al, 2016). Pol II promoters provide flexibility in terms of spatial and temporal control of expression in vivo, and they are more likely to produce long transcripts, since Pol II is not hampered by the presence of short internal termination sites, as is the case for Pol III (Arimbasseri et al, 2013).…”

Section: Targeted Mutagenesis Of Genes In M Truncatula Using Direct mentioning

confidence: 99%

“…The remaining three vectors produce a single transcript with gRNA cassettes separated by 20-bp Csy4 hairpins (Tsai et al, 2014), 77 bp tRNA Gly genes , or 15-bp ribozyme cleavage sites (in addition to a synthetic ribozyme at the 39 end of the transcript) (Haseloff and Gerlach, 1988;Tang et al, 2016). Thanks to the advantages over Pol III promoters mentioned above, we chose to use a Pol II promoter to drive the expression of the polycistronic gRNAs.…”

Section: Targeted Mutagenesis Of Genes In M Truncatula Using Direct mentioning

confidence: 99%

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A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

et al. 2017

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We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

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Section: Discussioncontrasting

confidence: 59%

Section: Targeted Mutagenesis Of Genes In M Truncatula Using Direct mentioning

confidence: 99%

Section: Targeted Mutagenesis Of Genes In M Truncatula Using Direct mentioning

confidence: 99%

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A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

et al. 2017

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“…Furthermore, the development and customization of plant CRISPR/Cas9 toolboxes or platforms result in more powerful molecular tools for fundamental research and crop breeding (Lowder et al ., 2015; Ma et al ., 2015; Mao et al ., 2016; Tang et al ., 2016; Wang et al ., 2015; Xie et al ., 2015; Xing et al ., 2014). Rice is the most frequently used crop species for testing and applying CRISPR‐Cas9 tools.…”

Section: Introductionmentioning

confidence: 99%

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Qin

et al. 2017

Plant Biotechnology Journal

231

152

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Summary CRISPR‐Cpf1 is a newly identified CRISPR‐Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR‐Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR‐Cpf1. We found that pre‐crRNAs with a full‐length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs. In addition, the specificity and transmission of the mutation were investigated, and the behaviours of crRNA‐Cpf1‐induced plant targeted genome mutagenesis were assessed. Taken together, our results indicate that CRISPR‐Cpf1 expression via stable transformation can efficiently generate specific and heritable targeted mutations in rice and thereby constitutes a novel and important approach to specific and precise plant genome editing.

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“…1B). This approach was validated in rice where it led to the same mutagenesis frequencies as the conventional twopromoters system [84].…”

Section: Expression and Addressingmentioning

confidence: 99%

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Collonnier

Guyon‐Debast

Maclot

et al. 2017

Methods

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a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

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A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants

Cited by 152 publications

References 11 publications

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

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