A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants
Tomas Cermak,Shaun J. Curtin,Javier Gil-Humanes,Radim Cegan,Thomas J. Y. Kono,Eva Konečná,Joseph J. Belanto,Colby G. Starker,Jade W. Mathre,Rebecca L. Greenstein,Daniel F. Voytas +10 more
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TLDR
An integrated reagent toolkit and streamlined protocols work across diverse plant species to enable sophisticated genome edits and it is demonstrated that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons.Abstract:
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 25-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)read more
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Posted ContentDOI
CRISPRi-based circuits for genetic computation in plants
Muhammad Adil Khan,Gabrielle Herring,Marina Núñez Oliva,Elliott Fourie,Jia-Yuan Zhu,Benjamin Johnston,Jahnvi Pflüger,Tessa Swain,Christiane Pflüger,James P. B. Lloyd,David Secco,Ian Small,Brendan N. Kidd,Ryan Lister +13 more
TL;DR: This work created a range of engineered repressible promoters of different strengths and used them as integrators for the construction of NOT and NOR gates in Arabidopsis cells, providing a new platform for sophisticated and deliberate spatio-temporal control of gene expression in plants.
Journal ArticleDOI
Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
TL;DR: In this paper , up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors in rice, peanut, chickpea, and cowpea.
Book ChapterDOI
Double strand break (DSB) repair pathways in plants and their application in genome engineering
TL;DR: This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what the authors have learned over the last decades about them.
Book ChapterDOI
Design and Assembly of CRISPR/Cas9 Reagents for Gene Knockout, Targeted Insertion, and Replacement in Wheat.
Tomas Cermak,Shaun J. Curtin +1 more
TL;DR: This protocol demonstrates the construction of reagents using a comprehensive genome engineering kit to create single and multiple gene "knockingouts," site-specific chromosome deletions and gene replacement or "knockins" including the use of geminivirus replicons (GVRs).
Journal ArticleDOI
Transcriptional and post-transcriptional controls for tuning gene expression in plants.
TL;DR: In this paper , a review of gene expression in the model plants Arabidopsis thaliana and Nicotiana benthamiana with an eye toward control points that may be used to predictably modify gene expression.
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Multiplex Genome Engineering Using CRISPR/Cas Systems
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