TALEN‐mediated targeted mutagenesis produces a large variety of heritable mutations in rice

Abstract

SummaryCRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALENinduced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations.

TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol

“…This mutation rate compares very favorably with earlier reported TALEN mediated targeted mutagenesis ranging from 4 to 31 % (Shan et al 2013; Wendt et al 2013; Zhang et al 2013, 2016). The observed high mutation rate can be a result of the TALEN scaffold, target genes, and target region.…”

Applications and roles of the CRISPR system in genome editing of plants

“…The TALEN-induced mutations were also stably transmitted to the T1 and T2 populations by normal Mendelian inheritance. Their results contrast with other published report for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3 % of the overall mutations (Zhang et al 2016b). …”

“…The CRISPR/Cas9 system represents dramatic scientific progress that allows the development of more complex useful agronomic traits through synthetic oligonucleotides (Belhaj et al 2013;Quetier 2016). Genome editing based on the CRISPR/Cas9 system has been successfully applied in dozens of diverse plant species, including the sweet orange (Jia and Wang 2014a), arabidopsis (Gao et al 2015b;Jiang et al 2013;Li et al 2014), tobacco (Gao et al 2015a, b;Jiang et al 2013;Li et al 2014;Lowder et al 2015), maize (Basak and Nithin 2015;Liang et al 2014;Puchta and Fauser 2013;Schuster et al 2015;Svitashev et al 2015;Xing et al 2014), rice (Mikami et al 2015a, b;Wang et al 2015;Xu et al 2014;Zhang et al 2016b), and wheat (Basak and Nithin 2015;Shan et al 2014;Sugano et al 2014;Upadhyay et al 2013;Wang et al 2014).…”

“…Basak and Nithin 2015;Duan et al 2015;Jameson and Song 2015;Jiang et al 2013;Lowder et al 2015;Ma et al 2015; Mikami et al 2015a, b;Schiml et al 2014;Seo et al 2015;Shan et al 2014;Sugano et al 2014;Wang et al 2015;Woo et al 2015;Xie et al 2015;Xie and Yang 2013;Xu et al 2014;Zhang et al 2016b;Zhang et al 2014; Zhou et al Gao et al 2015a;Li et al 2014; Upadhyay et al Ali et al 2015;Jiang et al 2013;Johnson et al 2015;Lowder et al 2015;Nekrasov et al 2013;Upadhyay et al 2013) MaizeZea mays ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4 Liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), acetolactate synthase (ALS) genes (ALS1 and ALS2)(Basak and Nithin 2015; Liang et al 2014; Puchta and Fauser 2013; Schuster et al 2015; Svitashev et al 2015; Xing et al Basak and Nithin 2015; Shan et al 2014; Sugano et al 2014; Upadhyay et al 2013; Wang et al 2014) Soybean Glycine max Green fluorescent protein (GFP) transgene GmPDS11GmPDS18(Du et al 2016;Jacobs et al 2015;Michno et al 2015;Ricroch and Henard-Damave 2015) Potato Solanum tuberosum Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4) Phytophthora infestans resistance genes(Butler et al 2015;Richter et al 2012; Schaart et al glucuronidase (GUS) transgene(Michno et al 2015) …”

Genome Editing in Plants: An Overview of Tools and Applications

“…These include creation of point mutations, insertion of new genes in specific locations or deletion of large regions of the nucleotide sequences, and correction or substitution of individual genetic elements and gene fragments [4,6,10,20,23,44,73]. While introducing modifications to various genomic elements in plant cells and examining the results, scientists were able to investigate the role of individual genes in the functioning of individual cells and the organism as a whole.…”