Repurposing of Anthocyanin Biosynthesis for Plant Transformation and Genome Editing
Ontology highlight
ABSTRACT:
SUBMITTER: He Y
PROVIDER: S-EPMC8525376 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Editing bacterial genomes is an essential tool in research and synthetic biology applications. Here, we describe multiplex genome editing by natural transformation (MuGENT), a method for accelerated evolution based on the cotransformation of unlinked genetic markers in naturally competent microorganisms. We found that natural cotransformation allows scarless genome editing at unprecedented frequencies of ?50%. Using DNA substrates with randomized nucleotides, we found no evidence for bias during natural cotransformation, indicating that this method can be used for directed evolution studies. Furthermore, we found that natural cotransformation is an effective method for multiplex genome editing. Because MuGENT does not require selection at edited loci in cis, output mutant pools are highly complex, and strains may have any number and combination of the multiplexed genome edits. We demonstrate the utility of this technique in metabolic and phenotypic engineering by optimizing natural transformation in Vibrio cholerae. This was accomplished by combinatorially editing the genome via gene deletions and promoter replacements and by tuning translation initiation of five genes involved in the process of natural competence and transformation. MuGENT allowed for the generation of a complex mutant pool in 1 wk and resulted in the selection of a genetically edited strain with a 30-fold improvement in natural transformation. We also demonstrate the efficacy of this technique in Streptococcus pneumoniae and highlight the potential for MuGENT to be used in multiplex genetic interaction analysis. Thus, MuGENT is a broadly applicable platform for accelerated evolution and genetic interaction studies in diverse naturally competent species.
| S-EPMC4066482 | biostudies-literature
Project description:A mutant allele of the transcription factor gene MYB10 from apple induces anthocyanin production throughout the plant. This gene, including its upstream promoter, gene coding region and terminator sequence, was introduced into apple, strawberry and potato plants to determine whether it could be used as a visible selectable marker for plant transformation as an alternative to chemically selectable markers, such as kanamycin resistance. After transformation, red coloured calli, red shoots and red well-growing plants were scored. Red and green shoots were harvested from apple explants and examined for the presence of the MYB10 gene by PCR analysis. Red shoots of apple explants always contained the MYB10 gene but not all MYB10 containing shoots were red. Strawberry plants transformed with the MYB10 gene showed anthocyanin accumulation in leaves and roots. No visible accumulation of anthocyanin could be observed in potato plants grown in vitro, even the ones carrying the MYB10 gene. However, acid methanol extracts of potato shoots or roots carrying the MYB10 gene contained up to four times higher anthocyanin content than control plants. Therefore anthocyanin production as result of the apple MYB10 gene can be used as a selectable marker for apple, strawberry and potato transformation, replacing kanamycin resistance.
| S-EPMC3210953 | biostudies-literature
Project description:Plantation forestry of Eucalyptus urophylla × Eucalyptus grandis supplies high-quality raw material for pulp, paper, wood, and energy and thereby reduces the pressures on native forests and their associated biodiversity. Nevertheless, owing to the heterozygosity of the E. urophylla × E. grandis genetic background, germplasm improvement by crossbreeding tends to be inefficient. As an alternative approach, genetic engineering of Eucalyptus can be used to effectively improve germplasm resources. From a strategic standpoint, increasing the plantation productivity and wood quality by transgenic technology has become increasingly important for forest industry. In this study, we established a fluorescence labelling method using CRISPR/Cas9 technology to obtain positive transformed progenies. The positive transformed progenies were easily obtained from the genetically modified population via fluorescence screening. This system can be used as a plant genome site-specific editing tool and may be useful for improving Eucalyptus genetic resources.
| S-EPMC8143419 | biostudies-literature
Project description:Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT-ATG reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T0 plants, pPE2-generated mutants occurred with 0%-31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT-ATG reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.
| S-EPMC7747961 | biostudies-literature
Project description:Plants have been explored for many years as inexpensive and versatile platforms for the generation of vaccines and other biopharmaceuticals. Plant viruses have also been engineered to either express subunit vaccines or act as epitope presentation systems. Both icosahedral and helical, filamentous-shaped plant viruses have been used for these purposes. More recently, plant viruses have been utilized as nanoparticles to transport drugs and active molecules into cancer cells. The following review describes the use of both icosahedral and helical plant viruses in a variety of new functions against cancer. The review illustrates the breadth of variation among different plant virus nanoparticles and how this impacts the immune response.
| S-EPMC5874652 | biostudies-other
Project description:Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.
| S-EPMC7589382 | biostudies-literature
Project description:CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.
| S-EPMC8747350 | biostudies-literature
Project description:Cassava starch is a widely used raw material for industrial production. South Chinese cassava cultivar 8 (Manihot esculenta Crantz cv. SC8) is one of the main locally planted cultivars. In this study, an efficient transformation system for cassava SC8 mediated with Agrobacterium strain LBA4404 was presented for the first time. Cassava friable embryogenic calli (FECs) were transformed through the binary vector pCAMBIA1304 harboring GUS- and GFP-fused genes driven by the CaMV35S promoter. The transformation efficiency was increased in the conditions of Agrobacterium strain cell infection density (OD600 = 0.65), 250 µM acetosyringone induction, and agro-cultivation with wet FECs for 3 days in dark. Based on the optimized transformation protocol, approximately 120-140 independent transgenic lines per mL settled cell volume (SCV) of FECs were created by gene transformation in approximately 5 months, and 45.83% homozygous mono-allelic mutations of the MePDS gene with a YAO promoter-driven CRISPR/Cas9 system were generated. This study will open a more functional avenue for the genetic improvement of cassava SC8.
| S-EPMC9498335 | biostudies-literature
Project description:Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1-crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.
| S-EPMC5316869 | biostudies-literature
Project description:With the advances in genomic sequencing, many organisms with novel biological properties are ripe for use as emerging model organisms. However, to make full use of them, transformation methods need to be developed to permit genome editing. Here, we present the development of transformation for the fungus fly Bradysia (Sciara) coprophila; this may serve as a paradigm for the development of transformation for other emerging systems, especially insects. Bradysia (Sciara) has a variety of unique biological features, including locus-specific developmentally regulated DNA amplification, chromosome imprinting, a monopolar spindle in male meiosis I, non-disjunction of the X chromosome in male meiosis II, X chromosome elimination in early embryogenesis, germ-line-limited (L) chromosomes and high resistance to radiation. Mining the unique biology of Bradysia (Sciara) requires a transformation system to test mutations of DNA sequences that may play roles for these features. We describe a Bradysia (Sciara) transformation system using a modified piggyBac transformation vector and detailed protocols we have developed to accommodate Bradysia (Sciara) specific requirements. This advance will provide a platform for us and others in the growing Bradysia (Sciara) community to take advantage of this unique biological system. In addition, the versatile piggyBac vectors described here and transformation methods will be useful for other emerging model systems.
| S-EPMC9323590 | biostudies-literature