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).
Bibliographical noteFunding Information:
We thank members of the Voytas lab for helpful discussions and insights. We particularly thank Lynn Hu for technical assistance, L?zaro Peres (Universidade de S?o Paulo, Brazil) and Agustin Zs?g?n (Universidade Federal de Vi?osa, Brazil) for input on target genes in tomato, Joseph Guhlin (College ofBiological Sciences, University ofMinnesota) for inputon M. truncatula NCR target genes, Aaron Hummel for providing the plasmids with structurally optimized gRNA scaffolds, Evan Ellison for the GmUbi promoter reagents, and Kit Leffler for help with the figures. This work was funded by grants fromthe National Science Foundation (IOS-1339209 and IOS-1444511) and the Department of Energy (DE-SC0008769). S.J.C. was supported by the laboratory of Nevin D. Young under National Science Foundation Award IOS-1237993.