Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion

Shimatani, Zenpei; Kashojiya, Sachiko; Takayama, M.; Terada, Rie; Arazoe, Takayuki; Ishii, Hisaki; Teramura, Hiroshi; Yamamoto, Tsuyoshi; Komatsu, Hiroki; Miura, Kenji; Ezura, Hiroshi; Nishida, Keiji; Ariizumi, Tohru; Kondo, Akihiko

doi:10.1038/nbt.3833

Cited by 638 publications

(412 citation statements)

References 17 publications

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“…These systems have been applied to mammalian cells, animals and plants 13–18 . Other CRISPR-guided BE systems using human AID (activation-induced cytidine deaminase) 19 or AID ortholog PmCDA1 from sea lamprey 20 have been reported and demonstrated in eukaryotic organisms 21,22 . Some researchers used BE3 to engineer premature coding termination to provide an alternative approach to knock out genes 15,18,23 .…”

Section: Introductionmentioning

confidence: 99%

Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion

Zheng

Wang

et al. 2018

Commun Biol

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The ability to precisely edit individual bases of bacterial genomes would accelerate the investigation of the function of genes. Here we utilized a nickase Cas9-cytidine deaminase fusion protein to direct the conversion of cytosine to thymine within prokaryotic cells, resulting in high mutagenesis frequencies in Escherichia coli and Brucella melitensis. Our study suggests that CRISPR/Cas9-guided base-editing is a viable alternative approach to generate mutant bacterial strains.

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Section: Introductionmentioning

confidence: 99%

Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion

Zheng

Wang

et al. 2018

Commun Biol

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“…Increased specificity has been achieved using paired TALEN or CRISPR/Cas9 nickases , truncated gRNAs (Fu et al, 2014), or dimeric CRISPR/Cas9 nucleases (Guilinger et al, 2014;Tsai et al, 2014). Other diverse applications have emerged, including targeted regulation of gene expression, targeted epigenetic modification, or site-specific base editing through deamination (La Russa and Qi, 2015;Kungulovski and Jeltsch, 2016;Zong et al, 2017;Shimatani et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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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“…The recent demonstrations of efficient base editing in crops (Lu and Zhu, 2017; Shimatani et al, 2017; Zong et al, 2017) (Figure 2A) provide a new method to select for and fine-tune desirable crop traits which are attributable to point mutations, such as 14 key agronomic traits in rice (Huang et al, 2010) and the flavor of tomatoes (Tieman et al, 2017). This can be further extended to other agricultural demands including improving nutrition and production of crops (reviewed in (Baltes et al, 2017)).…”

Section: Section 2: Applications Of Base Editingmentioning

confidence: 99%

“…This can be further extended to other agricultural demands including improving nutrition and production of crops (reviewed in (Baltes et al, 2017)). In rice, multiplex base editing of ALS and FTIP1e generated double mutants resistant to two herbicides (Shimatani et al, 2017). Interestingly, despite using similar Target-AID constructs, the rate of indels has been significantly higher in crops (Lu and Zhu, 2017; Shimatani et al, 2017) (Table 1) than in yeast or mammalian cells (Nishida et al, 2016).…”

Section: Section 2: Applications Of Base Editingmentioning

confidence: 99%

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

et al. 2017

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The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double stranded breaks and donor templates), and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

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Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion

Cited by 638 publications

References 17 publications

Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion

Highly efficient base editing in bacteria using a Cas9-cytidine deaminase fusion

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

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