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Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9
Abstract
The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.
... To investigate the role of Trx-like proteins in vivo, we first attempted to generate A. thaliana mutant plant lines deficient in TrxL2.1, TrxL2.2, and both ACHT1 and ACHT2, utilizing CRISPR/Cas9-mediated genome editing (33). Consequently, we obtained two TrxL2.1-deficient ...
... To simultaneously target two different sites in the plant genome, two single-guide RNA expression cassettes were integrated into one plasmid using the primers FH41, FH42, FH254, and FH255 (SI Appendix, Fig. S3A and Table S1). Plant transformation and screening were conducted as described by Hahn et al. (33). The trxl2.1(1), trxl2.1 (2), and acht lines were generated using the Col-0 line as a background. . ...
Significance
Plants modulate photosynthesis activity in response to the surrounding environment. It is well known that the redox-responsive protein thioredoxin (Trx) activates photosynthesis-related enzymes in the light. However, the factors involved in deactivating them are not well understood. Recent in vitro experiments suggest that several Trx and Trx-like proteins serve as oxidation factors for Trx-targeted proteins; thus, we examined their functions in vivo. Consequently, we found that f -type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx, were involved in oxidative deactivation of photosynthesis-related enzymes (e.g., fructose-1,6-bisphosphatase, Rubisco activase, and the ATP synthase γ-subunit). Thus, this study reveals the functions of oxidation factors in vivo and elucidates the regulation system for photosynthesis in the dark.
... The construction of a CRISPR/Cas9-mediated cpsf30 mutant was carried out following established protocols. 21 Custom single guide RNA (sgRNA) sequences targeting the AtCPSF30 (AT1G30460) gene were designed and cloned into the AtU6-26-sgRNA-SK vector (Table S1). The resulting sgRNA expression cassette was subsequently amplified and integrated into the Agrobacterium binary vector pCAMBIA1300-pYAO: Cas9 to generate the final transgene construct for plant transformation. ...
CPSF30, a key polyadenylation factor, also serves as an m⁶A reader, playing a crucial role in determining RNA fate post-transcription. While its homologs mammals are known to be vital for viral replication and immune evasion, the full scope of CPSF30 in plant, particular in viral regulation, remains less explored. Our study demonstrates that CPSF30 significantly facilitates the infection of turnip mosaic virus (TuMV) in Arabidopsis thaliana, as evidenced by infection experiments on the engineered cpsf30 mutant. Among the two isoforms, CPSF30-L, which were characterized with m⁶A binding activity, emerged as the primary isoform responding to TuMV infection. Analysis of m⁶A components revealed potential involvement of the m⁶A machinery in regulating TuMV infection. In contrast, CPSF30-S exhibited distinct subcellular localization, coalescing with P-body markers (AtDCP1 and AtDCP2) in cytoplasmic granules, suggesting divergent regulatory mechanisms between the isoforms. Furthermore, comprehensive mRNA-Seq and miRNA-Seq analysis of Col-0 and cpsf30 mutants revealed global transcriptional reprogramming, highlighting CPSF30’s role in selectively modulating gene expression during TuMV infection. In conclusion, this research underscores CPSF30’s critical role in the TuMV lifecycle and sets the stage for further exploration of its function in plant viral regulation.
... The proPYE::GUS line was previously described by [11]. To obtain olv loss of function mutants, the genome sequence was edited at the OLV locus by Crispr/Cas9 system according to published procedures [36]. sgRNAs were designed using the CRISPR-P2.0 ...
Iron (Fe) is a crucial micronutrient needed in many metabolic processes. To balance needs and potential toxicity, plants control the amount of Fe they take up and allocate to leaves and seeds during their development. One important regulator of this process is POPEYE (PYE). PYE is a Fe deficiency-induced key bHLH transcription factor (TF) for allocation of internal Fe in plants. In the absence of PYE, there is altered Fe translocation and plants develop a leaf chlorosis. NICOTIANAMINE SYNTHASE4 (NAS4), FERRIC-REDUCTION OXIDASE3 (FRO3), and ZINC-INDUCED FACILITATOR1 (ZIF1) genes are expressed at higher level in pye-1 indicating that PYE represses these genes. PYE activity is controlled in a yet unknown manner. Here, we show that a small Fe deficiency-induced protein OLIVIA (OLV) can interact with PYE. OLV has a conserved C-terminal motif, that we named TGIYY. Through deletion mapping, we pinpointed that OLV TGIYY and several regions of PYE can be involved in the protein interaction. An OLV overexpressing (OX) mutant line exhibited an enhanced NAS4 gene expression. This was a mild Fe deficiency response phenotype that was related to PYE function. Leaf rosettes of olv mutants remained smaller than those of wild type, indicating that OLV promotes plant growth. Taken together, our study identified a small protein OLV as a candidate that may connect aspects of Fe homeostasis with regulation of leaf growth.
... The sgRNAs were designed in such a way that they targeted an exon of GOI and preferably no off-target sequence with maximum mismatches number 3 (http://www.rgenome.net/casoffinder/). Primers (Table S1; 1/2 for At2g24610, 7/8 for At2g25220, 14/15 and 17/18 for At2g25970) were annealed, and the annealed primers were ligated (50-ng PFH6, 1-μL T 4 ligase, 2-μL T 4 buffer, 1.16-ng sgRNA, and H 2 O up to 20 μL) into Bbs1-digested plasmid PFH6 (GenBank accession number KY080689; Hahn et al., 2017). ...
Because of their photosynthetic capacity, leaves function as solar panels providing the basis for the growth of the entire plant. Although the molecular mechanisms of leaf development have been well studied in model dicot and monocot species, a lot of information is still needed about the interplay of the genes that regulate cell division and differentiation and thereby affect the photosynthetic performance of the leaf. We were specifically interested in understanding the differentiation of mesophyll and bundle sheath cells in Arabidopsis thaliana and aimed to identify genes that are involved in determining bundle sheath anatomy. To this end, we established a forward genetic screen by using ethyl methanesulfonate (EMS) for mutagenizing a reporter line expressing a chloroplast‐targeted green fluorescent protein (sGFP) under the control of a bundle sheath‐specific promoter. Based on the GFP fluorescence phenotype, numerous mutants were produced, and by pursuing a mapping‐by‐sequencing approach, the genomic segments containing mutated candidate genes were identified. One of the lines with an enhanced GFP fluorescence phenotype (named ELEVATED BUNDLE SHEATH CELLS SIGNAL 1 [ebss1] ) was selected for further study, and the responsible gene was verified by CRISPR/Cas9‐based mutagenesis of candidate genes located in the mapped genomic segment. The verified gene, At2g25970, encodes a K homology (KH) domain‐containing protein.
... Depending upon, how intrusive foreign DNA reacts, the CRISPR-Cas9-based system's mode of action can be categorised into three primary phases. The first phase is the procurement stage, which involves recognising foreign DNA and inserting a spacer sequence into the CRISPR array based on the target site in the DNA (Hahn et al., 2017). Second, the Cas9 protein is produced and the CRISPR array is translated into a precursor RNA transcript during the expression step (precrRNA). ...
The acceptance of new crop varieties by consumers is contingent on the presence of consumer-preferred traits, which include sensory attributes, nutritional value, industrial products and bioactive compounds production. Recent developments in genome editing technologies provide novel insight to identify gene functions and improve the various qualitative and quantitative traits of commercial importance in plants. Various conventional as well as advanced gene-mutagenesis techniques such as physical and chemical mutagenesis, CRISPR-Cas9, Cas12 and base editors are used for the trait improvement in crops. To meet consumer demand, breakthrough biotechnologies, especially CRISPR-Cas have received a fair share of scientific and industrial interest, particularly in plant genome editing. CRISPR-Cas is a versatile tool that can be used to knock out, replace and knock-in the desired gene fragments at targeted locations in the genome, resulting in heritable mutations of interest. This review highlights the existing literature and recent developments in CRISPR-Cas technologies (base editing, prime editing, multiplex gene editing, epigenome editing, gene delivery methods) for reliable and precise gene editing in plants. This review also discusses the potential of gene editing exhibited in crops for the improvement of consumer-demanded traits such as higher nutritional value, colour, texture, aroma/flavour, and production of industrial products such as biofuel, fibre, rubber and pharmaceuticals. In addition, the bottlenecks and challenges associated with gene editing system, such as off targeting, ploidy level and the ability to edit organelle genome have also been discussed.
... Another option is performing fluorescence in situ hybridization (FISH) or colorimetric in situ hybridization (CISH) using the mRNA sequence of the biomarker as a probe (Femino et al., 1998;Marcino, 2013). Finally, functional validation of differentially expressed genes and cell type function can be investigated through loss-of-function mutants or overexpression lines (Capecchi, 1989;Visscher et al., 2015;Hahn et al., 2017). ...
Over the last decades, research on postembryonic root development has been facilitated by “omics” technologies. Among these technologies, microarrays first, and RNA sequencing (RNA-seq) later, have provided transcriptional information on the underlying molecular processes establishing the basis of System Biology studies in roots. Cell fate specification and development have been widely studied in the primary root, which involved the identification of many cell type transcriptomes and the reconstruction of gene regulatory networks (GRN). The study of lateral root (LR) development has not been an exception. However, the molecular mechanisms regulating cell fate specification during LR formation remain largely unexplored. Recently, single-cell RNA-seq (scRNA-seq) studies have addressed the specification of tissues from stem cells in the primary root. scRNA-seq studies are anticipated to be a useful approach to decipher cell fate specification and patterning during LR formation. In this review, we address the different scRNA-seq strategies used both in plants and animals and how we could take advantage of scRNA-seq to unravel new regulatory mechanisms and reconstruct GRN. In addition, we discuss how to integrate scRNA-seq results with previous RNA-seq datasets and GRN. We also address relevant findings obtained through single-cell based studies and how LR developmental studies could be facilitated by scRNA-seq approaches and subsequent GRN inference. The use of single-cell approaches to investigate LR formation could help to decipher fundamental biological mechanisms such as cell memory, synchronization, polarization, or pluripotency.
... Previous knock-out and knock-in experiments were restricted to model organisms, but nowadays experimental validation of candidate genes can be supported via knock-out and knockin experiments, using novel genome editing tools (e.g., Shalem et al., 2014;Hahn et al., 2017;Ueta et al., 2017). Genome-editing tools are already available for a broad range of taxa, including dozens of crop species, but developing a working system in non-model organisms can still be a difficult task that can take several months or even years to accomplish (Shan et al., 2020), so doing collaborative studies alongside experimental researchers is recommended. ...
In the last decade, genomics and the related fields of transcriptomics and epigenomics have revolutionized the study of the domestication process in plants and animals, leading to new discoveries and new unresolved questions. Given that some domesticated taxa have been more studied than others, the extent of genomic data can range from vast to nonexistent, depending on the domesticated taxon of interest. This review is meant as a rough guide for students and academics that want to start a domestication research project using modern genomic tools, as well as for researchers already conducting domestication studies that are interested in following a genomic approach and looking for alternate strategies (cheaper or more efficient) and future directions. We summarize the theoretical and technical background needed to carry out domestication genomics, starting from the acquisition of a reference genome and genome assembly, to the sampling design for population genomics, paleogenomics, transcriptomics, epigenomics and experimental validation of domestication-related genes. We also describe some examples of the aforementioned approaches and the relevant discoveries they made to understand the domestication of the studied taxa.
A set of peroxidases detoxifies H2O2 and mediates H2O2-dependent signal propagation. The peroxidases include peroxiredoxins, glutathione peroxidases, ascorbate peroxidases, and catalases. This at least partial redundancy impedes addressing individual proteins in living plant cells so that the protein functions are often studied by biochemical assays in vitro. In vivo analysis frequently relies on transgenic insertion lines resulting in the knockdown or knockout of the protein of interest. However, many proteins have multiple isoforms in close genomic arrangement so that even crossing of transgenic lines does not allow for a knockdown of all isoforms. The genes encoding for the three cytosolic peroxiredoxins PRXIIB, C, and D in Arabidopsis thaliana are located in close vicinity on chromosome 1 so that crossing over between the genes most rarely occurs and successful crossing of the plants appears impossible. Genome editing instead allows targeting of multiple isoforms and knocks out several genes at once. This chapter describes how to inactivate the three cytosolic peroxiredoxins by CRISPR/Cas9 in A. thaliana.
To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH–Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin–Benson cycle enzymes, NADP–malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is exceptionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chloroplast redox-regulatory system operates in vivo.
Reactive carbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are highly reactive, unwanted side-products of cellular metabolism maintained at harmless intracellular levels by specific scavenging mechanisms. MGO and GO are metabolized through the glyoxalase (GLX) system, which consists of two enzymes acting in sequence, GLXI and GLXII. While plant genomes encode a number of different GLX isoforms, their specific functions and how they arose during evolution are unclear. Here, we used Arabidopsis (Arabidopsis thaliana) as a model species to investigate the evolutionary history of GLXI and GLXII in plants and whether the GLX system can protect plant cells from the toxicity of RCS other than MGO and GO. We show that plants possess two GLX systems of different evolutionary origins and with distinct structural and functional properties. The first system is shared by all eukaryotes, scavenges MGO and GO, especially during seedling establishment, and features Zn2+-type GLXI proteins with a metal co-factor preference that were present in the last eukaryotic common ancestor. GLXI and GLXII of the second system, featuring Ni2+-type GLXI, were acquired by the last common ancestor of Viridiplantae through horizontal gene transfer from proteobacteria and can together metabolize keto-D-glucose (KDG, glucosone), a glucose-derived RCS, to D-gluconate. When plants displaying loss-of-function of a Viridiplantae-specific GLXI were grown in KDG, D-gluconate levels were reduced to 10-15% of those in the wild type, while KDG levels showed an increase of 48-67%. In contrast to bacterial GLXI homologs, which are active as dimers, plant Ni2+-type GLXI proteins contain a domain duplication, are active as monomers, and have a modified second active site. The acquisition and neofunctionalization of a structurally, biochemically, and functionally distinct GLX system indicates that Viridiplantae are under strong selection to detoxify diverse RCS.
C 4 photosynthesis has independently and convergently evolved approx. 70 times in flowering plants. Most C 4 plant species display a distinct leaf anatomy (Kranz‐anatomy) and similar metabolic and biochemical features. Computational modeling indicates that C 4 photosynthesis evolves from C 3 photosynthesis via C 3 –C 4 intermediate states [1,2]. C 3 –C 4 intermediacy evolves via simple loss of function mutations and causes a nitrogen disbalance between leaf mesophyll and bundle sheath cells. Overcoming this nitrogen disbalance requires the shuttling of organic acids between mesophyll and bundle sheath cells, which under permissive conditions drives the system towards C 4 photosynthesis [3]. Genetic hybrids between C 3 and C 3 –C 4 intermediate species segregate for subtraits of C 3 –C 4 intermediacy and thereby enable the identification of key enablers by quantitative genetics. Identification of such enables is a prerequisite for engineering of the C 4 trait into C 3 species by synthetic biology approaches [4].
We have recently identified such enablers. These include cis‐regulatory elements in the promoters of genes encoding components of photorespiration. To experimentally test these enablers in a C 3 genetic background and to eventually recapitulate the evolutionary trajectory from C 3 to C 4 photosynthesis in the model plant A. thaliana , we have developed a non‐invasive visual marker that enables detection of Cas9‐activity in planta [5,6]. We have used this maker system to test different concepts for homology‐based gene editing and have developed a reliable system for homology‐directed repair in Arabidopsis [7]. These protocol will now be employed for introduction of C 4 ‐enablers into Arabidopsis.
Support or Funding Information
We appreciate support by the Deutsche Forschungsgemeinschaft (EXC 1028, IRTG 1525, ERA‐CAPS project C4BREED).
This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Significance
Plant chloroplasts have acquired an evolutionary “redox switch” that regulates the activities of photosynthesis-related enzymes in response to a dynamically changing environment. Because the metabolic state in chloroplasts drastically fluctuates in response to changes in the surrounding environment, it has been considered essential to adjust the activities of chloroplast enzymes. Many efforts have been made to elucidate this regulation in detail. Its physiological role is thought to be due to the fact that fine-tuning of the activity is essential for efficient photosynthesis, but it is still unclear how this is really the case. We have shown that the redox switch of NADP-malate dehydrogenase plays a crucial role in the optimal growth of plants under fluctuating light conditions and prolonged darkness.
The CRISPR/Cas9 system enables precision editing of the genome of the model plant Arabidopsis thaliana and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in Arabidopsis would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed Cas9 gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing in vivo: BIALAPHOS RESISTANCE (BAR) and GLABROUS1 (GL1). BAR confers resistance to glufosinate and is widely used as a positive selection marker; GL1 is required for the formation of trichomes. Reversion of a frameshift null BAR allele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of the BAR gene through the germline. We hypothesize that few BAR revertant cells in a highly chimeric plant likely provide system-wide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting the GL1 gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting of GL1 is suitable for assessing and optimizing Cas9-mediated gene-editing in Arabidopsis.
The production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damages in an error-prone way. In case of radiation, multiple double strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site directed nucleases (SDNs), also referred to as sequence specific nucleases (SSNs), like the CRISPR/Cas system, enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at predefined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish it from natural variants. This applies to changes induced by DSBs, as well as single strand breaks (SSBs), and involves repair by Non-Homologous End-Joining (NHEJ) and Homologous Recombination (HR). The recent development of SDN-based "DNA-free" approaches makes the discrimination between mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of the new SDN technologies, much faster and more precise genome editing becomes available at reasonable costs and potentially not requiring time consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in context of the regulatory situation for genetically modified crop plants. This article is protected by copyright. All rights reserved.
The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development.
The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers’ ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.
The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by site-specific nucleases to initiate DNA repair reactions that are either based on non-homologous end joining (NHEJ) or homologous recombination (HR). Recently, the CRISPR/Cas system emerged as the most important tool for genome engineering due to its simple structure and its applicability to a wide range of organisms. Here, we review the current status of its various applications in plants, where it is used for the successful generation of stable mutations in a steadily growing number of species through NHEJ. Furthermore, tremendous progress in plant genome engineering by HR was obtained by the setup of replicon mediated and in planta gene targeting techniques. Finally, other complex approaches that rely on the induction of more than one DNA lesion at a time such as paired nickases to avoid off-site effects or controlled genomic deletions are beginning to be applied routinely.
Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently-developed tool for the introduction of site-specific double stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments.
Copyright © 2014. Published by Elsevier Inc.
Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code - enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Engineered nucleases can be used to induce site-specific double strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5% up to 70.0%. Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions.This article is protected by copyright. All rights reserved.
Significance
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been used to generate targeted gene editing in plants. However, it is not known whether CRISPR/Cas-induced gene modifications in plants occur in somatic cells only or whether some or all of the modifications can enter the germ line to become heritable. Through systematic and multigenerational analysis, this study demonstrates that although the majority of gene modifications detected in the first generation CRISPR/Cas transgenic Arabidopsis plants were somatic mutations only, heritable mutations could be found in subsequent generations. In addition, deep sequencing of CRISPR/Cas-modified Arabidopsis genomes did not detect any off-targets. The work demonstrates that the CRISPR/Cas method can effectively create specific gene modifications in planta that are stably transmitted through the germ line to future generations.
The Type II CRISPR/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants but are limited by off-target mutations. Here we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif (PAM) sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website.
Cas-OFFinder free access at http://cas-offinder.snu.ac.kr CONTACT: baesau@snu.ac.kr (SB); jskim01@snu.ac.kr (J-SK).
Ditching Invading DNA
Bacteria and archaea protect themselves from invasive foreign nucleic acids through an RNA-mediated adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas). Jinek et al. (p. 816 , published online 28 June; see the Perspective by Brouns ) found that for the type II CRISPR/Cas system, the CRISPR RNA (crRNA) as well as the trans-activating crRNA—which is known to be involved in the pre-crRNA processing—were both required to direct the Cas9 endonuclease to cleave the invading target DNA. Furthermore, engineered RNA molecules were able to program the Cas9 endonuclease to cleave specific DNA sequences to generate double-stranded DNA breaks.
We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.
To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.
We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.
The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks (DSBs) at preselected genomic sites that are repaired by non-homologous end-joining (NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas-mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR-based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off-target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable to that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.This article is protected by copyright. All rights reserved.
http://www.genome.arizona.edu/crispr/index.html
Precision genome engineering using CRISPR/Cas holds great promise for disease modelling, precision medicine and basic research. There, a combination of an RNA guided enzyme (Cas) and a target homologous single guide RNA (sgRNA) is employed to introduce precise genetic alterations or perturbations. For all applications of genome engineering such as genetic screens or mutation modelling accuracy and efficacy of engineering constructs are of high importance. To aid scientists in selecting the best targeting constructs upfront, we developed computational methods for the fast and accurate identification of sgRNA target sites. Here we present E-CRISP, a well-established and far developed web-service for designing sgRNAs against 56 organisms in a gene-by-gene fashion (e-crisp.org). It provides detailed efficacy and specificity assessments for each sgRNA target alongside detailed information about the targeted locus. E-CRISP offers a wide range of features and options not provided in other tools while also delivering sensible defaults to aid users through the design process. To specifically design high-complex sgRNA libraries for use in high throughput genetic screens, we further present the CRISPR Library Designer (CLD, github.com/boutroslab/cld), an integrated bioinformatics application for the design of custom sgRNA libraries for all organisms with annotated genomes. Using CLD, constructs can be designed for applications using a large variety of Cas9 variants (CRISPRn, CRISPRa/CRISPRi, Cpf1) and targeting any specific region of an organism’s genome. We validated the productivity of CLD by designing a dedicated library of 12,471 sgRNAs and performed a pooled loss-of-function screen identifying all essential components of the TNF-related apoptosis inducing ligand (TRAIL) pathway. Our results confirm that CLD selects a very high fraction of efficient sgRNAs, increasing the rate of efficient gene editing and reducing library size. Together, E-CRISP and CLD constitute an effective toolkit for the design and evaluation of genome engineering constructs across various scales, experimental techniques and organisms.
Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22 nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome editing events, the mutation efficiency at these target sites was estimated to be 3 - 8%. In addition, off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome editing efficiency than the perfectly matched site. Further analysis suggests that mis-match position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.
TheAgrobacteriumvacuum infiltration method has made it possible to transformArabidopsis thalianawithout plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containingAgrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, andAgrobacteriumcould be applied to plants at a range of cell densities. Repeated application ofAgrobacteriumimproved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation ofArabidopsisfor efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.
INTRODUCTION
This procedure describes a quick miniprep for plant DNA isolation. It yields a small quantity of poorly purified DNA. However, many samples can be processed in a short time. When used with Arabidopsis , the method generally provides DNA of sufficient quality for polymerase chain reaction (PCR) amplification of cleaved amplified polymorphic sequences (CAPS) and simple sequence length polymorphism (SSLP) markers. Repeated freeze-thaw cycles should be avoided, because they result in samples that will no longer work as PCR templates.
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm
include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log‐expectation score, and refinement using tree‐dependent
restricted partitioning. The speed and accuracy of MUSCLE are compared with T‐Coffee, MAFFT and CLUSTALW on four test sets
of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest,
rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable
from T‐Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences
of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely
available at http://www.drive5. com/muscle.
Multigeneration analysis reveals the inheritance
- Z Feng
- Y Mao
- N Xu
- B Zhang
- P Wei
- D L Yang
- Z Wang
- Z Zhang
- R Zheng
- L Yang
- L Zeng
- X Liu
- J K Zhu
Feng, Z., Mao, Y., Xu, N., Zhang, B., Wei, P., Yang, D. L., Wang, Z., Zhang, Z., Zheng, R., Yang,
L., Zeng, L., Liu, X. and Zhu, J. K. (2014). Multigeneration analysis reveals the inheritance,
www.bio-protocol.org/e2384
Application of the CRISPR-Cas system for efficient genome engineering in plants
- Y Mao
- H Zhang
- N Xu
- B Zhang
- F Gou
- J K Zhu
Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F. and Zhu, J. K. (2013). Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6(6): 2008-2011.
Interference by clustered regularly interspaced 20
- E Semenova
- M M Jore
- K A Datsenko
- A Semenova
- E R Westra
- B Wanner
- J Van Der Oost
- S J Brouns
- K Severinov
Semenova, E., Jore, M. M., Datsenko, K. A., Semenova, A., Westra, E. R., Wanner, B., van der
Oost, J., Brouns, S. J. and Severinov, K. (2011). Interference by clustered regularly interspaced
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