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Title: A multi-purpose toolkit to enable advanced genome engineering in plants

Here, 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 twelve 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.more » The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).« less
 [1] ;  [2] ;  [3] ;  [4] ;  [5] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Genetics, Cell Biology & Development and Center for Genome Engineering
  2. Univ. of Minnesota, St. Paul, MN (United States). Dept. of Plant Pathology and Dept. of Agronomy & Plant Genetics; USDA-ARS Cereal Disease Lab., St. Paul, MN (United States)
  3. Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Genetics, Cell Biology & Development and Center for Genome Engineering; Calyxt Inc., New Brighton, MN (United States)
  4. Inst. of Biophysics of Chinese Academy of Sciences, Brno (Czech Republic). Dept. of Plant Developmental Genetics
  5. Univ. of Minnesota, St. Paul, MN (United States). Dept. of Agronomy & Plant Genetics
Publication Date:
Grant/Contract Number:
sc0008769; IOS-1339209; IOS-1444511; IOS-1237993
Accepted Manuscript
Journal Name:
Plant Cell
Additional Journal Information:
Journal Volume: 29; Journal Issue: 6; Journal ID: ISSN 1040-4651
American Society of Plant Biologists
Research Org:
Donald Danforth Plant Science Center, St. Louis, MO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division
Country of Publication:
United States
OSTI Identifier: