CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry
TL;DR: A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots and the new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies.
Abstract: To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T0) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies. A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots. The new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies.
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01 Nov 2003
TL;DR: Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways.
Abstract: Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene (PDS3) encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarf phenotypes of the pds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in the pds3 mutant. Further analysis showed that high level of phytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype of the pds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.
216 citations
TL;DR: In this paper , a protocol for in vitro polyploid induction of highbush blueberry (Vaccinium corymbosum L.) leaf tissues was studied by using different concentrations of colchicine and oryzalin.
Abstract: Polyploid induction is of utmost importance in horticultural plants for the development of new varieties with desirable morphological and physiological traits. Polyploidy may occur naturally due to the formation of unreduced gametes or can be artificially induced by doubling the number of chromosomes in somatic cells. In this experiment, a protocol for in vitro polyploid induction of highbush blueberry (Vaccinium corymbosum L.) leaf tissues was studied by using different concentrations of colchicine and oryzalin. Oryzalin was found to be highly toxic to this species, while the adventitious shoot organogenesis media enriched with 25 and 250 µM colchicine was able to induce polyploidization, with significant differences among the treatments used. Higher concentrations of both antimitotic agents led to the browning and death of the leaf tissues. The polyploids obtained showed several morphological differences when compared with the diploid shoots. Flow cytometry analysis was used to confirm the ploidy level of the regenerated shoots, demonstrating that a total of 15 tetraploids and 34 mixoploids were obtained. The stomatal sizes (length and width) of the tetraploids were larger than those of the diploids, but a reduced stomatal density was observed as compared to the controls. These shoots will be acclimatized and grown until they reach the reproductive phase in order to test their potential appeal as new varieties or their use for breeding and genetic improvement.
6 citations
TL;DR: In this paper , the use of CRISPR/Cas9 was applied to highbush blueberry (Vaccinium corymbosum L) using Agrobacterium-mediated transformation.
Abstract: Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry “Berkeley”, and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.
2 citations
TL;DR: In this article , a review of modern advances in genome editing of horticultural plants is presented, where the main ways of delivering genetic constructs to plants as well as limitations that complicate the editing of target genes are considered.
Abstract: Horticultural plants, in particular fruit trees, berry crops, and ornamentals, are valuable objects for studying their genetic and biochemical properties. Along with traditional methods of studying these plants, modern molecular genetic technologies are emerging, in particular genome editing using CRISPR/Cas9 nucleases. In this review, we have analyzed modern advances in genome editing of horticultural plants. To date, it has become possible to improve many plant characteristics using this technology, e.g., making plants resistant to biotic and abiotic stress factors, changing the time of flowering and fruit ripening, changing the growth characteristics of plants, as well as the taste properties of their fruits. CRISPR/Cas9 genome editing has been successfully carried out for many horticultural plants. Dozens of genes from these plants have been modified by means of genome editing technology. We have considered the main ways of delivering genetic constructs to plants as well as limitations that complicate the editing of target genes. The article reviews the prospects of using genome editing to improve the valuable properties of plants important to humans.
1 citations
TL;DR: In this paper , a transgenic tobacco line carrying a marker, beta-glucuronidase gene (gusA), was created and a CRISPR-Cas9 editing vector was introduced to knock out gusA.
Abstract: Chimeric editing is often reported in gene editing. To assess how the general chimeric editing is, we created a transgenic tobacco line carrying a marker, beta-glucuronidase gene (gusA), introduced a CRISPR-Cas9 editing vector into the transgenic tobacco line for knocking out gusA, and then investigated the gusA editing efficiencies in T0 and subsequent generations. The editing vector carried a Cas9 gene, which was driven by the cauliflower mosaic virus 35S promoter, and two guide RNAs, gRNA1 and gRNA2, which were driven by Arabidopsis U6 (AtU6) and U3 (AtU3) promoter, respectively. The two gRNAs were designed to knock out a 42-nucleotide fragment of the coding region of gusA. The editing vector was transformed into gusA-containing tobacco leaves using Agrobacterium tumefaciens-mediated transformation and hygromycin selection. Hygromycin-resistant, independent T0 transgenic lines were used to evaluate gusA-editing efficiencies through histochemical GUS assays, polymerase chain reactions (PCR), and next-generation sequencing of PCR amplicons. Profiles of targeted sequences of 94 T0 transgenic lines revealed that these lines were regenerated from non-edited cells where subsequent editing occurred and created chimeric-edited cells in these lines during or after regeneration. Two of them had the target fragment of 42 bp pairs of nucleotides removed. Detail analysis showed that on-target mutations at the AtU6-gRNA1 site and the AtU3-gRNA2 site were found in 4.3% and 77.7% of T0 transgenic lines, respectively. To overcome the issue of extremely low editing efficiencies in T0 lines, we conducted a second round of shoot induction from the chimeric line(s) to enhance the success of obtaining lines with all or most cells edited. The mutation profiles in T0 transgenic lines provide valuable information to understand gene editing in plant cells with constitutively expressed CRISPR-Cas9 and gRNAs.
1 citations
References
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TL;DR: GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage, and Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Abstract: We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
9,765 citations
Broad Institute1, Commonwealth Scientific and Industrial Research Organisation2, Massachusetts Institute of Technology3, Hebrew University of Jerusalem4, Science for Life Laboratory5, Pittsburgh Supercomputing Center6, Oklahoma State University–Stillwater7, Griffith University8, University of Wisconsin-Madison9, Dresden University of Technology10, California Institute for Quantitative Biosciences11, Flanders Institute for Biotechnology12, Parco Tecnologico Padano13, United States Department of Agriculture14, Purdue University15, Indiana University16
TL;DR: This protocol provides a workflow for genome-independent transcriptome analysis leveraging the Trinity platform and presents Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes.
Abstract: De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.
6,369 citations
TL;DR: In this paper, the authors characterized Cpf1, a putative class 2 CRISPR effector, which is a single RNA-guided endonuclease lacking tracrRNA and utilizes a T-rich protospacer-adjacent motif.
3,436 citations
TL;DR: Recent progress is described in understanding of how cells detect and signal the presence and repair of one particularly important form of DNA damage induced by ionizing radiation—the DNA double-strand break (DSB).
Abstract: To ensure the high-fidelity transmission of genetic information, cells have evolved mechanisms to monitor genome integrity. Cells respond to DNA damage by activating a complex DNA-damage-response pathway that includes cell-cycle arrest, the transcriptional and post-transcriptional activation of a subset of genes including those associated with DNA repair, and, under some circumstances, the triggering of programmed cell death. An inability to respond properly to, or to repair, DNA damage leads to genetic instability, which in turn may enhance the rate of cancer development. Indeed, it is becoming increasingly clear that deficiencies in DNA-damage signaling and repair pathways are fundamental to the etiology of most, if not all, human cancers. Here we describe recent progress in our understanding of how cells detect and signal the presence and repair of one particularly important form of DNA damage induced by ionizing radiation-the DNA double-strand break (DSB). Moreover, we discuss how tumor suppressor proteins such as p53, ATM, Brca1 and Brca2 have been linked to such pathways, and how accumulating evidence is connecting deficiencies in cellular responses to DNA DSBs with tumorigenesis.
2,385 citations
TL;DR: The utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy is demonstrated.
Abstract: *Summary Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available ‘entry vector’ and are then recombined into various ‘destination vectors’ for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of ‘pEarleyGate’ plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.
1,688 citations