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

Journal Article · · The Plant Cell
DOI:https://doi.org/10.1105/tpc.16.00922· OSTI ID:1423880
 [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

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. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Research Organization:
Donald Danforth Plant Science Center, St. Louis, MO (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
sc0008769; IOS-1339209; IOS-1444511; IOS-1237993
OSTI ID:
1423880
Journal Information:
The Plant Cell, Vol. 29, Issue 6; ISSN 1040-4651
Publisher:
American Society of Plant BiologistsCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 323 works
Citation information provided by
Web of Science

Cited By (73)

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Editing the Medicago truncatula Genome: Targeted Mutagenesis Using the CRISPR-Cas9 Reagent book January 2018
Genome Editing in Potato with CRISPR/Cas9 book January 2019
CRISPR/Cas9-Mediated Gene Editing Tool and Fathomless Genetic and Metabolic Engineering Applications in Plants book January 2018
Advanced genetic tools enable synthetic biology in the oleaginous microalgae Nannochloropsis sp. journal March 2018
CRISPR/Cas-mediated gene targeting in plants: finally a turn for the better for homologous recombination journal January 2019
Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation journal February 2019
CRISPR-based genome editing in wheat: a comprehensive review and future prospects journal April 2019
Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants journal January 2020
CRISPR/Cas-mediated genome editing for crop improvement: current applications and future prospects journal December 2018
From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas journal August 2019
A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis journal November 2019
Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing journal January 2020
De novo domestication of wild tomato using genome editing journal October 2018
Domestication of wild tomato is accelerated by genome editing journal October 2018
A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants journal March 2018
Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome journal January 2019
Plant gene editing through de novo induction of meristems journal December 2019
Prime genome editing in rice and wheat journal March 2020
Targeted mutagenesis in wheat microspores using CRISPR/Cas9 journal April 2018
Facilitating gene editing in potato: a Single-Nucleotide Polymorphism (SNP) map of the Solanum tuberosum L. cv. Desiree genome journal February 2020
BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors journal May 2019
Does co-transcriptional regulation of alternative splicing mediate plant stress responses? journal February 2019
Gene editing in plants: progress and challenges journal January 2019
YSL3-mediated copper distribution is required for fertility, grain yield, and size in Brachypodium posted_content December 2019
Translocation and duplication from CRISPR-Cas9 editing in Arabidopsis thaliana posted_content August 2018
CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis posted_content June 2019
Building customizable auto-luminescent luciferase-based reporters in plants posted_content October 2019
Genomic Outcomes of Haploid Induction Crosses in Potato (Solanum tuberosum L.) posted_content October 2019
Using CRISPR/Cas9 genome editing in tomato to create a gibberellin-responsive dominant dwarf DELLA allele journal June 2018
Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing journal January 2019
Efficient induction of heritable inversions in plant genomes using the CRISPR /Cas system journal April 2019
Genetic Modification for Wheat Improvement: From Transgenesis to Genome Editing journal March 2019
How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis journal February 2020
Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines journal February 2020
A multiplex guide RNA expression system and its efficacy for plant genome engineering journal March 2020
SWISS: multiplexed orthogonal genome editing in plants with a Cas9 nickase and engineered CRISPR RNA scaffolds journal June 2020
Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis journal January 2019
CBF-dependent and CBF-independent regulatory pathways contribute to the differences in freezing tolerance and cold-regulated gene expression of two Arabidopsis ecotypes locally adapted to sites in Sweden and Italy journal December 2018
A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana journal June 2018
Genomic Outcomes of Haploid Induction Crosses in Potato ( Solanum tuberosum L.) journal December 2019
Efficient Multiplex Genome Editing Induces Precise, and Self-Ligated Type Mutations in Tomato Plants journal July 2018
Modifications in Organic Acid Profiles During Fruit Development and Ripening: Correlation or Causation? journal November 2018
Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing journal September 2019
Genome Editing in Plants: Exploration of Technological Advancements and Challenges journal November 2019
Cas Endonuclease Technology—A Quantum Leap in the Advancement of Barley and Wheat Genetic Engineering journal May 2019
TCR Affinity Biases Th Cell Differentiation by Regulating CD25, Eef1e1, and Gbp2 journal March 2019
Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation posted_content August 2018
Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis posted_content September 2018
A multiplex guide RNA expression system and its efficacy for plant genome engineering posted_content March 2020
From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas text January 2020
A multiplex guide RNA expression system and its efficacy for plant genome engineering posted_content March 2020
Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system text January 2019
Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering journal February 2019
A multiplex guide RNA expression system and its efficacy for plant genome engineering posted_content March 2020
A multiplex guide RNA expression system and its efficacy for plant genome engineering posted_content March 2020
CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines journal September 2017
A unified multi-kingdom Golden Gate cloning platform journal July 2019
Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat journal February 2018
In Planta Processing of the SpCas9–gRNA Complex journal October 2017
Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting posted_content January 2018
CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula journal December 2017
Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration journal January 2018
Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus journal April 2018
Characterization of a semidominant dwarfingPROCERAallele identified in a screen for CRISPR/Cas9-induced suppressors of loss-of-function alleles journal November 2018
Intron-Based Single Transcript Unit CRISPR Systems for Plant Genome Editing journal February 2020
Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases journal October 2017
Utilization of transcription factors for controlling floral morphogenesis in horticultural plants journal January 2018
Setaria Comes of Age: Meeting Report on the Second International Setaria Genetics Conference journal September 2017
Editing Citrus Genome via SaCas9/sgRNA System journal December 2017
A Review of the Interactions between Wheat and Wheat Pathogens: Zymoseptoria tritici, Fusarium spp. and Parastagonospora nodorum journal April 2018
CRISPRMatch: An Automatic Calculation and Visualization Tool for High-throughput CRISPR Genome-editing Data Analysis journal January 2018

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